Chemosphere 171 (2017) 427e434

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Column transport studies of 3-nitro-1,2,4-triazol-5-one (NTO) in soils
Noah Mark a, Jennifer Arthur a, Katerina Dontsova a, b, *, Mark Brusseau a, Susan Taylor c, Jirí Simunek da

Dept. of Soil, Water, and Environmental Science, Univ. of Arizona, 1177 E. Fourth St., PO Box 210038, Shantz Bldg. #38, Tucson, AZ 85721-0038, USA
Biosphere 2, University of Arizona, Marshall Building, Room 523, 845 N. Park Avenue, Tucson, AZ 85721-0158, USAc
Cold Regions Research and Engineering Laboratory, U.S. Army Engineer Research and Development Center, 72 Lyme Road, Hanover, NH 03755-1290, USAd
Dept of Environmental Sciences, Univ. of California Riverside, Riverside, CA 92521, USAbh i g h l i g h t s
 This is the ﬁrst study to examine transport behavior of dissolved NTO, a new IM compound.
 We measured adsorption, retardation and transformation of dissolved NTO in 8 soils.
 NTO absorption and retardation were low (Kd < 1 cm3 g1).
 Transformation rates increased with time for high OC soils.
 Organic carbon and microbial activity promoted NTO transformation.

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 25 October 2016
Received in revised form
15 December 2016
Accepted 15 December 2016
Available online 18 December 2016

Development of the new, insensitive, energetic compound, NTO (3-nitro-1,2,4-triazol-5-one), createsneed for the data on NTO's fate and transport to predict its behavior in the environment and potential forgroundwater contamination. To measure the transport of NTO in soils, we conducted miscibledisplacement experiments under steady state and interrupted ﬂow conditions using eight soils having
varying physical and geochemical properties. The breakthrough curve (BTC) data were analyzed using
temporal moment analysis and simulated using HYDRUS-1D to determine transport parameters andbetter understand the mechanisms of sorption and transformation. Parameters determined from themiscible-displacement study were compared to results obtained from batch experiments conducted forthe same soils, and examined in relation to soil properties. Column NTO linear adsorption coefﬁcients(Kd) were low and correlated well (P ¼ 0.000049) with measurements from the batch studies. NTO
transformation rate constants increased and NTO recovery decreased with increase in soil organic carbon(OC) content. Autoclaved soils had slower transformation rates and greater NTO recoveries indicatingthat microorganisms play a role in NTO transformation. In addition, the transformation rate increased
with time in soils with higher OC. Monod-type kinetics was implemented in HYDRUS-1D to simulate theobserved increase in transformation rate with time. We think this phenomenon is due to bacterial
growth. Results indicate very low adsorption of NTO in a range of soils, but natural attenuation throughtransformation that, depending on soil OC content and hydraulic residence time, could result in complete
removal of NTO.
© 2016 Elsevier Ltd. All rights reserved.

Handling Editor: T Cutright
Keywords:
NTO
Insensitive munitions
Energetic materials
Natural attenuation
Transport

1. Introduction

Abbreviations and acronyms: ATO, 5-amino-1,2,4-triazol-3-one; DNAN, 2,4dinitroanisole; IM, Insensitive Munition; NTO, 3-nitro-1,2,4-triazol-5-one.
* Corresponding author. Biosphere 2, University of Arizona, Marshall Building,Room 523, 845 N. Park Avenue, Tucson, AZ 85721-0158, USA.
E-mail address: dontsova@email.arizona.edu (K. Dontsova).
http://dx.doi.org/10.1016/j.chemosphere.2016.12.067
0045-6535/© 2016 Elsevier Ltd. All rights reserved.

NTO (3-nitro-1,2,4-triazol-5-one) is a new energetic compounddeveloped by the U.S. military for insensitive munitions (IMs). IMs
retain the performance of traditional explosives but are better able
to tolerate unplanned stimuli, such as mechanical shock and hightemperatures (Beyard, 2007). As a result of incomplete detonation,solid IM explosives containing NTO may be deposited on range

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N. Mark et al. / Chemosphere 171 (2017) 427e434

soils, and after dissolution (Taylor et al., 2013, 2015a, 2015b) canpotentially reach groundwater. Complex interaction of the dissolved IM components with soil constituents is key to understanding the threat of groundwater contamination. NTO is highly
soluble (16,642.0 mg L1 at 25  C) (Spear et al., 1989) and its
transport to groundwater used as potable water source could resultin human exposure to the chemical. While NTO showed low
toxicity to the freshwater organism Ceriodaphnia dubia, green algae
Selenastrum capricornutum (Haley et al., 2009), and Rana pipiens
tadpoles (Stanley et al., 2015), it is a male reproductive toxicant inrats (Wallace, 2011; Lent et al., 2015) and was reported to form
transformation products that can be toxic if they behave similarly
to metabolic intermediates of other nitro-compounds (Le Campion
et al., 1999b). Based on these risks, accurate soil adsorption coefﬁcients (Kds) and transformation rate constants are necessary tosimulate and predict NTO fate and transport to groundwater.
NTO's pKa is 3.7e3.76, and it is negatively charged at environmentally relevant pHs (Le Campion et al., 1997; Smith and Cliff,1999). Low estimated octanol-water partition coefﬁcients (Kow)(logKows 0.37e1.03) (Sokkalingam et al., 2008) suggest that NTO
would not strongly adsorb to soil organic matter through hydrophobic interactions and, therefore, be more mobile than othercomponents in IM mixtures, such as 2,4-dinitroanisole (DNAN)(logKows 1.58e1.92) (Sokkalingam et al., 2008; Hawari et al., 2015).
Microbial transformation studies have shown that NTO can undergo nitroreduction under anaerobic conditions (Krzmarzick et al.,2015) and form 5-amino-1,2,4-triazol-3-one (ATO). ATO can furthertransform under aerobic conditions to CO2, urea, and inorganic
nitrogen species (Le Campion et al., 1999b; Krzmarzick et al., 2015).
Mark et al. (2016) conducted a series of kinetic and equilibrium
batch experiments to determine ﬁrst order transformation rate
constants (k), Kds and Freundlich adsorption coefﬁcients (Kfs) for
NTO using multiple soils with a range of properties. NTO was
weakly adsorbed, with Kds ranging between 0.02 and 0.51 cm3 g1.
Soil afﬁnity for NTO was not inﬂuenced by OC content of the soil,
but was strongly affected by soil pH. Adsorption decreased as soil
pH increased, probably due to the inﬂuence of protonation on theinteraction between soil surfaces and NTO. This is supported bystudies with pure minerals (Linker et al., 2015) that showed thatNTO did not adsorb to negatively charged layer silicate clays, but
had higher afﬁnity for goethite that was positively charged. Estimates for NTO ks ranged between 0.0004 h1 and 0.0221 h1. There
was a signiﬁcant positive relationship between OC in soils andmeasured ks (Mark et al., 2016) and sterilized soils experienced
smaller mass loss than same soils without sterilization suggesting
microbial transformation, possibly with OC as the energy source.
To further elucidate processes involved in the reactive transportof NTO in soils, we conducted miscible-displacement experiments
using saturated repacked columns under steady state and transient
ﬂow conditions. NTO k and Kd were determined using temporal
moment analysis (TMA) and HYDRUS-1D solute transportmodeling software. Monod-type kinetics was implemented in
HYDRUS to evaluate the occurrence of temporally dependent NTO
transformation. To assess the impact of the experimental approach,transport parameters (k, transformation rate for Monod-type kinetics, and Kd) were compared to the results of the batch studies(Mark et al., 2016). This is the ﬁrst study to examine transport
behavior of dissolved NTO in soils.
2. Materials and methods
2.1. Soils
For the NTO transport experiments we selected eight uncontaminated surface soils (top 12 inches or 30 cm) with a wide rangeof properties collected (except for Catlin) on military installations
across the United States. They were a subset of eleven soils used for
NTO batch adsorption and transformation studies by Mark et al.
(2016). Some of these soils had been used previously in studies of
explosive and propellant formulation constituents (Dontsova et al.,2006, 2007, 2009a, 2009b; Taylor et al., 2012). As the same soils
were used in batch and saturated ﬂow studies we were able tocompare determined parameters to verify if the previously
observed processes were responsible for NTO attenuation. The
selected soils had mixed particle size, ranging from ﬁne (clay loam)
to coarse (loamy sand) and a wide range of pH (4.40e8.21) and OC
concentrations (0.34e5.28%). Their characteristics are summarizedin Table A1 and in Mark et al. (2016).
2.2. NTO solution preparation and quantiﬁcation
Technical grade NTO used in the experiments was obtained fromUS Army Armament Research, Development and Engineering
Center (ARDEC), Picatinny Arsenal. Calibration standards for NTO
were prepared using the same NTO. One mg L1 solutions wereprepared and NTO concentration was quantiﬁed using Dionex Ultimate 3000 high performance liquid chromatograph (HPLC)
equipped with a diode array detector (ThermoFisher, MA) as
described in Mark et al. (2016). The HPLC quantiﬁcation method
was adapted from Le Campion et al. (1999a). Mobile phase, acetonitrile (ACN): deionized water (75:25) with 0.1% TFA, was run isocractically at 1 mL min1. Oven temperature was set at 32  C. NTO
and its transformation products were separated using a Thermo
Scientiﬁc Hypercarb Column. NTO was detected at approximately
2.5 min using a 315 nm wavelength. The UV detector was set at
220 nm to monitor for presence of ATO (Le Campion et al., 1999a).
No ATO was detected in any analyzed samples.
2.3. Saturated ﬂow experiments
To measure the transport of NTO in soils, we conducted saturated miscible-displacement experiments under steady state andtransient conditions. Solutions of NTO were used as the source.
Concentrations of NTO, as well as a conservative tracer (bromide),used to characterize water ﬂow through the columns, were determined in the column efﬂuent and used to construct breakthroughcurves.
Small columns (7 cm length with 1.18 cm internal diameter glass
tubes with PTFE caps, Supelco, Bellefonte, PA) allowed us to minimize the amount of source material needed and reduce hazardous
waste produced in the study. Between 11 and 12 g of soil was
packed homogenously over a bottom layer of silanized glass woolthat prevented migration of particles. Average packed bulk density(r) (determined from the mass of air-dried soil used to pack acolumn of known volume) ranged between 1.71 ± 0.06 g cm3(Camp Guernsey soil) and 1.31 ± 0.02 g cm3 (Arnold AFB soil).
Saturated ﬂow parameters, water content (q) and packed bulk
density (r) for all studied soils are shown in Table B2.
A layer of glass wool overlaid the packed soil and helped toevenly distribute incoming ﬂow. To avoid air entrapment, columns
were saturated from the bottom up with the background solution(0.005M CaCl2) for approximately 1e1.5 h. After saturation, porevolume (PV) was determined by measuring the volume of solution
needed to saturate the packed column. Tygon microbore tubing
connected to a Cole-Parmer (Vernon Hills, IL) Master ﬂex peristaltic
pump was attached to the top of each column to supply 1 mg L1
NTO solution with 0.005M CaBr2 tracer at a given ﬂux. A
0.02 mL min1 ﬂow rate, or 1.1 cm h1 Darcy ﬂux, was used(measured average ﬂow rate was 0.0197 ± 0.0019 mL min1, orDarcy ﬂux of 1.083 ± 0.0017 cm h1). Efﬂuent was collected

N. Mark et al. / Chemosphere 171 (2017) 427e434

continuously into 4 mL amber vials using Teledyne ISCO (Lincoln,
NE) Foxy 200 Fraction collector with a 200-vial capacity. Pump wascalibrated for constant target ﬂow prior to the start of the experiment. Volumetric ﬂow rate was monitored based on collected
efﬂuent. After 4 to 17 PV, depending on the soil, inﬂow was
switched to 0.005M CaCl2 solution and the ﬂow continued forseveral more pore volumes to evaluate NTO desorption and elution
from the soil.
Flow interruption tests, wherein ﬂow was stopped for 24 h,
were performed for all soils to better characterize and quantify
rate-limited mass-transfer and transformation processes (Brusseau
et al., 1989, 1997). When ﬂow is stopped during the arrival wave
(NTO injection phase), mass-transfer and transformation processes
continue, which will be reﬂected in a decrease in concentration if
transformation or sorption is rate limited. These ﬂow interruption
experiments were repeated for sterilized Catlin and Sassafras soils(OC content of 5.28 and 1.30%, respectively) to determine whether
solute concentration loss was caused by microbial transformation.
Soils were sterilized by autoclaving three times over three days
with 1 h exposure to 120  C each time.
Bromide was analyzed using Ion Chromatography (Dionex ICS
5000 with conductivity detector). Efﬂuent solution samples wereﬁltered through 0.45 mm Millex-HV PVDF ﬁlter (EMD Millipore
Darmstadt, Germany), and placed in labeled 4-mL amber vials priorto HPLC analysis for NTO. The tracer allowed us to distinguish between non-equilibrium processes attributed to physical versus
chemical properties and to determine dispersion and diffusion in
the columns.
After the end of each experiment, the columns were divided into
thirds and weighed. Extractions were performed on each soil
sample to determine the amount of NTO remaining in the soil. A
mixture of soil and acetonitrile in 1:2 ratio was agitated for 24 h,centrifuged, ﬁltered through 0.45 mm Millex-HV PVDF ﬁlter (EMD
Millipore Darmstadt, Germany), and analyzed using HPLC (U. S.
Environmental Protection Agency, 2006).
2.4. Numerical modelingTemporal moment analysis (TMA) was used to analyze breakthrough curves. TMA described in Supplement appendix 1 allowed
to quantify solute transport properties such as dispersivity, massbalance ratio, or a ratio between mass of efﬂuent and inﬂow solute,and retardation factor R (equal to 1þ (r/q) Kd, where r is the soil
bulk density, Kd is the linear adsorption coefﬁcient, and q is thewater content).
 
HYDRUS-1D model (Simunek and van Genuchten, 2008; 
Simunek et al., 2016) was also used to analyze breakthroughcurves obtained from the column experiments. Constant pressure
head boundary conditions were used to model continuous ﬂow
experiments, while variable pressure head boundary conditions
were used to model ﬂow interruption tests.
Solute transport was modeled using the advection-dispersion
equation:

q

vC
vS
v2 C
vC
þ r ¼ qD 2  q
f
vt
vt
vz
vz

(1)

where C is the solution concentration [ML3], S is the sorbed concentration [MM1] (S ¼ Kd*C), D is the dispersion coefﬁcient which
includes both molecular diffusion and hydrodynamic dispersion[L2T1], q is the volumetric ﬂuid ﬂux density (speciﬁc discharge)[LT1] evaluated using the Darcy-Buckingham law, and f is a sinksource term which includes various zero and ﬁrst order reactions[ML3T1]. In this application, f represents the transformation(mass loss) of NTO. In some cases, this process was represented as a429

ﬁrst order reaction occurring only in the liquid phase and is deﬁned
as follows:f ¼ kqC(2)

Alternatively, we also used a modiﬁed version of HYDRUS-1D
that employs Monod kinetics to describe BTCs where efﬂuent
concentration decreases with time (De Wilde et al., 2009). Thismodel accounts for bacteria growth using the following kinetics:

dX
¼
dt



mm


C
X  kdecay X
Ks þ C(3)

where X is the biomass concentration [ML3], C is the liquid NTO
concentration [ML3], mm is the speciﬁc biomass growth rate coefﬁcient [T1], Ks is the half saturation constant [ML3], and kdecay isthe decay rate [T1]. The corresponding equation describing NTO
concentration is as follows:



dC
1
C
¼
X
mm
dt
Y
Ks þ C(4)

where Y is the yield [MM1].
In this model, bacterial growth requires the presence of NTO. If
the amount of bacteria remains constant, the NTO transformation
rate (m*X) stays the same and the BTC of NTO would attain steadystate conditions wherein the efﬂuent concentration of NTO remains
constant during continued constant injection. However, depending
on extant conditions, signiﬁcant bacterial growth may cause anincrease in the magnitude of NTO transformation, causing NTO
efﬂuent concentrations to decrease with time (e.g., Brusseau et al.,1999, 2006).
2.5. Parameter estimation
First, the TMA was used to analyze bromide breakthroughcurves to determine physical parameters characterizing the column experiments, namely l. Then, NTO BTC data were analyzed
using TMA to provide estimates for NTO transport parameters,
including l (cm), R, Kd (cm3 g1), and k (h1). Mass balance calculations were performed for NTO by mass balance ratio, r. NTO
mass balance estimates were compared to recovery of the conservative tracer.
In HYDRUS-1D simulations of bromide transport, l for the tracer
was optimized using the TMA value as the initial estimate. For the
NTO simulations, the HYDRUS-1D-optimized l obtained for the
tracer and Kd calculated by TMA for NTO were used as initial estimates for optimization by HYDRUS-1D. The transformation rate
coefﬁcients (ﬁrst order or Monod based) were also optimized.
R2 values characterize the correspondence between simulated
and measured solute concentrations and the conﬁdence intervals
for the optimized parameters show the behavior of the objectivefunction around its minimum. Parameter estimates were considered statistically signiﬁcant if conﬁdence intervals did not intersectwith zero. At least three experiments were conducted for each soil.
Interrupted and continuous ﬂow experiments were treated as
replicates for the purposes of statistical analysis for TMA estimatesand analyzed separately for HYDRUS-1D. Differences between
treatments were considered signiﬁcant if conﬁdence intervals did
not overlap at 95% probability. When multiple column experiments
were performed for the same soil under either continuous orinterrupted ﬂow conditions, their HYDRUS-1D parameter estimatesand conﬁdence intervals were averaged. The reaction parameters
determined using the inverse mode of HYDRUS-1D were comparedto the same parameters independently determined in soil batchstudies. Agreement between the two sets of values provides

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N. Mark et al. / Chemosphere 171 (2017) 427e434

conﬁdence that the determined parameters accurately characterize
the transport of NTO in soils.
3. Results and discussion
3.1. Conservative tracer
The mean l determined from the tracer BTC for both interrupted
and continuous ﬂow experiments was larger for coarser soils suchas Camp Butner and Camp Guernsey (0.28 ± 0.136 and0.26 ± 0.198 cm, respectively) than for the ﬁner Catlin and Camp
Swift soils (0.11 ± 0.151 and 0.17 ± 0.194 cm, respectively) (Tables B1
and B2), but differences were not signiﬁcant at a 95% conﬁdence
level. In general, the values were relatively small (many notsigniﬁcantly different from zero) as expected for short repacked

Table 1Mass recovered (%) in efﬂuent of column transport experiments involving NTO andconservative tracer, bromide. Soils are listed in order of decreasing OC content.
Soil

NTO

Bromide

Mean

SD

Mean

SD

CatlinArnold AFB
ButnerLimestone Hills
Sassafras
Camp Guernsey
Florence MRCamp Swift

34.2
45.0
52.7
64.2
68.3
85.9
88.6
90.4

12.4
11.1
2.3
12.9
12.3
8.3
1.6
1.7

103.8
97.4
98.0
94.2
107.0
101.5
99.9
98.4

10.8
2.9
5.9
8.6
12.4
5.6
5.8
4.5

columns. Breakthrough of the conservative tracer occurred at one
pore volume (~140 min), which indicates a lack of preferential ﬂow.
Air entrainment within the column was not observed. Tracer
breakthrough curves were plotted on a pore volume basis tocompare their behavior to that of NTO. After ﬂow interruption,
efﬂuent concentrations of bromide resumed at similar values,
indicating minimal impact of diffusive mass-transfer processes
associated with physically heterogeneous domains (soil aggregates,
dead-end porosity, etc). Mass balance calculations showed full recovery of the tracer (Table 1).

3.2. NTO
Fig. 1 presents breakthrough curves for Camp Guernsey and
Arnold AFB soils and Fig. 2 presents breakthrough curves for
Sassafras and Catlin soils. Solid triangles represent measured values
for NTO concentration for experiments with and without ﬂow
interruption, while lines indicate BTCs simulated by HYDRUS-1D(Figs. 1 and 2). Values for l determined by HYDRUS-1D for NTO
were relatively similar to bromide (Table B2). 11 out of 16 treatments (ﬂow interruption and continuous ﬂow experiments for eachsoil type) showed that 95% conﬁdence intervals for NTO and bromide dispersivity values overlapped. In the 5 instances where 95%
CI did not overlap, in 4 soils dispersivity estimates for NTO werehigher than that of bromide, and in Sassafras soil (continuous ﬂow)
they were lower.
NTO breakthrough was observed after breakthrough of theconservative tracer, suggesting NTO adsorption to the solid phase,but for all soils this delay (retardation) was small. Four out of eightFig. 1. Breakthrough curves for NTO in Camp Guernsey and Arnold AFB soils. Dashed grey vertical line indicates timing of 24-h ﬂow interruption (FI). Grey solid vertical line
indicates time when solution was switched back to saturating solution to observe desorption and elution of NTO.

N. Mark et al. / Chemosphere 171 (2017) 427e434

431

Fig. 2. Breakthrough curves for NTO in untreated and autoclaved Sassafras and Catlin soils. Dashed grey vertical line indicates timing of 24-h ﬂow interruption. Grey solid verticalline indicates time when solution was changed back to saturating solution to observe desorption phase of the isotherm.

soils, Sassafras, Camp Guernsey, Florence MR, and Camp Swift,
exhibited behavior consistent with adsorption and ﬁrst ordertransformation observed in the batch studies (Mark et al., 2016). For
example, for Camp Guernsey soil (Fig. 1a and b), there is very little
retardation observed consistent with low Kd measured in batchstudies (0.02 cm3 g1). Steady state concentrations after breakthrough are consistent with ﬁrst order transformation, as is thedecrease in efﬂuent NTO concentration after 24 h ﬂow interruption
and return to steady state observed for these soils (Figs. 1b and 2a).
However, in the other four soils, Catlin (Fig. 2a and b), Arnold
AFB (Fig. 1c and d), Camp Butner, and Limestone Hills, efﬂuent
concentrations did not attain steady state, but rather decreased
with time, indicating an increase in the transformation rate. They
also exhibited sharper decreases in NTO concentration following
ﬂow interruption, such that NTO could no longer be detected, andafter ﬂow was restarted concentrations of NTO did not increase.
Such changes in efﬂuent concentrations with time can be explainedby a change in conditions within the column. One possible mechanism is a change in redox state of the column as the experiment
progresses. As has been shown by Krzmarzick at al. (2015), NTO
reduction, a ﬁrst step in its transformation, is favored underanaerobic conditions. Anaerobic conditions are more likely todevelop as a result of microbial respiration when OC is present andthis is consistent with the fact that the temporal increase intransformation rates was observed only for soils with high OC
content (Table B1). However, we do not have a direct measure ofchange in oxygen concentration or redox potential of the efﬂuent. It
is also possible that microbial growth, stimulated by OC in the soil,
could directly affect NTO concentration if microorganisms cometabolize NTO with the OC. NTO transformation linked tobacterial growth is supported by our modeling results. Applying
Monod-type kinetics for Catlin (Fig. 2c and d), Arnold AFB (Fig. 1cand d), Camp Butner, and Limestone Hills soil experiments in
HYDRUS-1D generated R2 values close to 1 (Table 2).
Mass balance calculations showed that NTO recovery was lessthan a 100% (Table 1) and similar between continuous and interrupted ﬂow experiments (not shown) but decreased with increasein OC content of the soil (Table 1) consistent with positive correlation between k values and OC observed in batch studies (Market al., 2016). No NTO or transformation products were recoveredfrom soil by acetonitrile extraction at the end of the column experiments. Since experiments were continued in desorption phase
until no NTO was recovered in the efﬂuent, no water extractable
NTO remained in the soils.
Further evidence for a microbial contribution to NTO transformation comes from the results of experiments conducted withsterilized soils, which were compared to the non-sterilized soil data
in Fig. 2 for Catlin (5.28% OC) and Sassafras (1.30% OC). For untreated Sassafras soil, the BTCs exhibited constant NTO concentrations and decreases in concentration upon ﬂow interruption
consistent with ﬁrst order transformation. For autoclaved Sassafras
soil, the efﬂuent NTO concentration was similar to inﬂow concentration, and ﬂow interruption had no effect, indicating no transformation. For Catlin soil, which had a Monod-type transformation
behavior and no recovery after ﬂow interruption in untreated columns, the efﬂuent concentrations were constant and concentrations recovered after ﬂow interruption for the sterilized treatments.
The k values (Table 3) were higher in untreated soils: k determined for untreated Sassafras soil was approximately 600 times
higher than k determined for sterilized Sassafras soil (which was

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N. Mark et al. / Chemosphere 171 (2017) 427e434

Table 2
NTO fate and transport parameters from temporal moment analysis (TMA) and HYDRUS-1D, linear adsorption coefﬁcients, Kd, ﬁrst order transformation rates, k, and initial
Monod transformation rate, with average rate shown in parentheses. In all cases, TMA estimates of k were determined for the portion of the BTC where steady state inﬂuent
concentration was achieved. TMA results include data from both interrupted ﬂow (FI) and continuous ﬂow experiments. Interrupted and continuous ﬂow experiments weretreated as replicates for the purposes of statistical analysis. HYDRUS-1D results show interrupted ﬂow (FI) and continuous ﬂow experiments separately. NA indicates that
results are not available. Soils are listed in order of decreasing OC content. CI ¼ 95% conﬁdence interval.
Soil

TMA

HYDRUS-1D

Kd, cm3 g1

CatlinCatlin FI
Arnold AFB
Arnold AFB FICamp Butner
Camp Butner FI
Limestone Hills
Limestone Hills FI
Sassafras
Sassafras FICamp GuernseyCamp Guernsey FI
Florence MR
Florence MR FICamp SwiftCamp Swift FIk, h1R

Kd, cm3 g1

Est.

CI

Est.

CI

Est.

0.33

0.12

1.70

0.25

NA

CI

0.39

0.16

1.99

0.38

NA

0.47

0.19

2.81

1.43

NA

0.22

0.14

1.61

0.40

NA

0.62

0.41

3.01

1.41

0.006

0.006

0.12

0.07

1.58

0.27

0.020

0.005

0.07

0.03

1.30

0.16

0.023

0.007

0.02

0.05

1.07

0.19

0.049

0.008

not signiﬁcantly different from zero), and the average Monodtransformation rate in untreated Catlin soil was approximately 3
times higher than the k value determined for columns using sterilized Catlin soil (Table 3). No transformation products wereobserved, and no NTO was recovered in soil by acetonitrile
extraction. Between the soils, transformation rates were higher for
the Catlin soil with or without sterilization.
Similarly to what was observed in batch studies (Mark et al.,2016), autoclaved and untreated soils also showed a signiﬁcantdifference in mass loss, particularly for soil with higher OC content,suggesting biotransformation (Fig. 2 and Table 3). Forty four
percent less NTO was recovered in the column efﬂuent in untreated
Catlin soil compared to recovery from columns using Catlin soil
sterilized by autoclave (24 vs 68%) (Table 3). Similarly smaller massbalance ratios were determined for autoclaved compared to untreated Sassafras soils (81% and 91%, respectively). Combined evidence from impact of sterilization on DNAN transformation rates,
mass loss, and shape of breakthrough curves, and from ability of
Monod rates to describe change in NTO concentrations for high OC
soils strongly supports importance of microbial activity for NTO
transformation.
Good ﬁts for BTC for Sassafras, Camp Guernsey, Florence MR,
and Camp Swift were achieved using an equilibrium adsorption
model suggesting that kinetic sorption was likely negligible(Table 2). With the exception of Florence MR and Camp Guernsey
soils, the Kd values determined from HYDRUS-1D matched wellwith TMA derived values and fell within 95% conﬁdence intervalsk, h1

Est.

CI

0.15
0.04
0.17
0.13
0.12
0.06
0.0001
0.15
0.05
0.40
0.03
0.05
0.03
0.04
2.55E-06
0.04

0.04
0.04
0.01
0.02
0.01
0.01
0.01
0.01
0.01
0.31
0.01
0.01
0.02
0.01
2.55E-06
0.01

Est.

0.100
0.076
0.009
0.064
0.065
0.041
0.040
0.009

Monod Trans. rate, h1
CI

Est.

CI

0.058 (0.123)
0.046 (0.133)
0.072 (0.121)
0.067 (0.133)
0.207 (0.259)
0.100 (0.183)
0.038 (0.154)
0.034 (0.186)

0.015
0.016
0.008
0.010
0.024
0.011
0.010
0.009

0.008
0.058
0.009
0.010
0.013
0.003
0.008
0.004

R2

0.97
0.96
0.99
0.98
0.99
0.99
0.99
0.99
0.99
0.96
0.98
0.98
0.98
0.97
0.98
0.99

(Table 2). Irreversible attenuation and transformation, as indicatedby k, was statistically signiﬁcant in TMA and when modeled by
HYDRUS-1D. The k values determined from HYDRUS-1D weregenerally in the same order of magnitude as TMA values but did not
fall within 95% conﬁdence intervals, with the exception of Florence
MR FI and Camp Swift FI (Table 2). Soils that had higher OC content,
Catlin, Arnold AFB, Camp Butner, and Limestone Hills, showed nonﬁrst order, irreversible attenuation as indicated by lower NTO recovery in efﬂuent and changes in transformation rates with time,
which was modeled by HYDRUS-1D using Monod-type kinetics.
Obtained transformation rate constants for these soils could not becompared directly to the TMA estimates.
There was a general agreement between batch and column
determined sorption parameters. Figures B1a and B1b show acomparison of NTO Kds determined from batch and column experiments using TMA and HYDRUS-1D. Correlation between Kds
derived from TMA BTC data and measured in batch experiments
was highly signiﬁcant (P ¼ 0.00020) (Figure B1a). The slope of theregression was higher than but not signiﬁcantly different from 1.
Kds derived from HYDRUS-1D using BTC data were also signiﬁcantly
correlated to, but lower than, Kd estimates in batch experiments(Figure B1b). Correlating an average of TMA and HYDRUS-1D
derived Kd versus batch Kd for the eight soils tested (Fig. 3a)yields a slope of regression equal to 0.97 (lower and upper 99% CI,
0.58 and 1.35, respectively, and P ¼ 0.000049) indicating that theestimates are the same. This increases our conﬁdence in thedetermined values, and as a result, in our ability to predict NTO

Table 3
Average NTO mass recovered (%) in the efﬂuent and the HYDRUS-1D-calculated transformation rate constant for column transport experiments involving untreated andsterilized Catlin and Sassafras soils. Average Monod transformation rate constant (h1) (an average of the NTO transformation rate constants using the initial and ﬁnal biomass
concentration, X) is shown for Catlin FI Untreated, while ﬁrst order transformation rate constant (h1) is shown for the remaining treatments. FI ¼ ﬂow interruption for 24 h.
Transformation rate constant, h1

Soil

Mass Recovery
Estimate

95% CI

Catlin FI Untreated
Catlin FI Sterilized
Sassafras FI Untreated
Sassafras FI Sterilized

24
68
81
91

17
45
77
90

a

Average Monod transformation rate (h1).

31
90
85
92

Estimate

95% CI

0.137a0.048
0.030
4.99E-05

0.128
0.038
0.025
1.76E-03

R2

0.147
0.059
0.036
1.86E-03

0.95
0.95
0.97
0.96

N. Mark et al. / Chemosphere 171 (2017) 427e434

433

The estimates of transformation rate constants, a combinationof ﬁrst order rates for soils with steady state conditions and mean
(0.1060 ± 0.0339 h1) or initial (0.0641 ± 0.0228 h1) Monod rates
for soils that did not, in column experiments were, on average,
more than an order of magnitude higher than ones determined in
the batch studies for the same soils (0.0043 ± 0.0033 h1). If wecompared only soils that had steady state rates we saw similar
trends: for batch studies rates equaled 0.0024 ± 0.0035 h1 and forcolumn studies 0.0505 ± 0.0221 h1. Correlation between batchand HYDRUS-1D determined values was weak (Fig. 3b). It was not
signiﬁcant when the intercept was allowed to vary but signiﬁcant(P ¼ 0.031698) if the intercept was set to zero. Obtained transformation rate constants also correlated with percent OC(P ¼ 0.003306) but also only with a zero intercept (Fig. 3c). A
positive relationship between OC and k values was observed inbatch studies (Mark et al., 2016). A slope of the correlation between
k estimates using batch and column experiments was signiﬁcantly
different from one.
We need to understand the large difference between transformation rates observed in column and batch experiments topredict NTO behavior in the environment based on measurements
done in the lab. One factor inﬂuencing the comparison of batch andcolumn biodegradation rate coefﬁcients is that the NTO supply isquite different. In a batch system the NTO is a ﬁxed source that
depletes with time. Conversely, for column experiments, there is aconstant supply of NTO. This maintains a maximal rate of transformation, and also allows for greater microbial growth. We would
expect that column experiments are closer to the ﬁeld conditions inthis respect and therefore provide a more realistic estimate of NTO
transformation.
This work emphasized the importance of microbial transformation for NTO fate in soils. It showed that conditions that are
favorable for microbial activity, such as high OC content, promote
NTO transformation, main mechanism of natural attenuation forthis compound that is highly soluble and negatively charged underconditions usually encountered in soils, and therefore weakly
adsorbed. These ﬁndings also provide a potential mechanism fortreatment of NTO-impacted soils.
4. Conclusion

Fig. 3. Correlation of NTO linear adsorption coefﬁcients (Kd) between batch measurements (Mark et al., 2016) and column measurements derived from the temporal
moment analysis and HYDRUS-1D (P ¼ 0.000049) (a). Correlation between batch(Mark et al., 2016) and column (derived from HYDRUS-1D (b, P ¼ 0.031698)) NTO
transformation rate constants; and between OC percent in the soil and column
transformation rate constant (c, P ¼ 0.003306). Values from continuous and ﬂow
interrupted experiments were averaged.

We conducted a series of saturated ﬂow experiments with andwithout ﬂow interruption (for 24 h equilibration period) for eightsoils collected on military ranges across the US and having a rangeof soil properties. Sorption Kds obtained from the column experiments were consistent with those determined from prior batchstudies. Conversely, larger transformation rate constants were obtained for the column study. NTO transformation rates increased
with increase in soil OC, as was observed for the batch study. Soils
with higher OC content also exhibited Monod kinetics of transformation characterized by increase in transformation rate withtime caused by bacterial growth.
Overall, the results indicate that NTO exhibits generally low
sorption. This would mean that NTO has a great potential for
advective transport in the subsurface. However, the results also
show that NTO is subject to signiﬁcant mass loss via biologicaltransformation. Depending upon extant site conditions, thistransformation may cause signiﬁcant attenuation during transport.
The results of this study may be useful for evaluating the signiﬁcance of attenuation and potential impacts on groundwater quality.
Acknowledgments

adsorption in other soils if we know their pH, a relationshipdeveloped by Mark et al. (2016) using batch study results.

This work was funded by the Strategic Environmental Researchand Development Program (SERDP), project ER-2220. We are434

N. Mark et al. / Chemosphere 171 (2017) 427e434

grateful to Anthony Di Stasio and Erika Rivera, US Army Armament
Research, Development and Engineering Center (ARDEC), Picatinny
Arsenal for providing NTO; Bonnie M. Packer, Rosa Gwinn, Lisa
DeGrazia, Bethany Keller, Jessica Milose, Amibeth Sheridan, Laurie
Stenberg, Sarah Gettier, Army National Guard Environmental
Directorate and URS Corporation, Germantown, MD and Mike
Heitmann, CH2M HILL Englewood, CO for collecting soils used in
the experiments on Army National Guard installations.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.chemosphere.2016.12.067.
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