The use of amino compounds for binding 2,4,6-trinitrotoluene in water

Article abstract of DOI:10.1016/S0269-7491(00)00077-4

Sites polluted with 2,4,6-trinitrotoluene (TNT) constitute a worldwide problem. In this work, chemical reactions for binding TNT to amino-compounds are proposed as an initial step for developing new remediation techniques to clean-up groundwater and soils contaminated with TNT. Indeed, addition of aniline and an amino acid-like cysteine caused a decrease in free TNT of 86 percent and 68-100 percent, respectively. Using 13C-NMR spectroscopy, it was shown that TNT chemically forms a Meisenheimer complex with cysteine and aniline in 1/1 (by vol.) H2O/d6-acetone.

Full text of DOI:10.1016/S0269-7491(00)00077-4

Environmental Pollution 111 (2001) 503±507
www.elsevier.com/locate/envpol
The use of amino compounds for binding 2,4,6-trinitrotoluene
in water
F. Fant a, A. De Sloovere b, K. Matthijsen b, C. Marle b, S. El Fantroussi b,
W. Verstraete b,*
aBioNMR and Peptide Synthesis, Department of Organic Chemistry, University of Gent, Krijgslaan 281 S4bis, B-9000 Gent, Belgium
bLaboratory of Microbial Ecology and Technology, University of Gent, Coupure Links 653, B-9000 Gent, Belgium
Received 2 September 1999; accepted 28 January 2000
``Capsule'': Addition of aniline and an amino acid-like cysteine to water decreased free TNT.
Abstract
Sites polluted with 2,4,6-trinitrotoluene (TNT) constitute a worldwide problem. In this work, chemical reactions for binding TNT
to amino-compounds are proposed as an initial step for developing new remediation techniques to clean-up groundwater and soils
contaminated with TNT. Indeed, addition of aniline and an amino acid-like cysteine caused a decrease in free TNT of 86% and 68±
100%, respectively. Using 13C-NMR spectroscopy, it was shown that TNT chemically forms a Meisenheimer complex with cysteine
and aniline in 1/1 (by vol.) H2O/d6-acetone. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: TNT; Amino acid; Meisenheimer complex
1. Introduction
Devliegher, 1997). In this study, a chemically based
reaction for binding TNT to amino-compounds, such as
cysteine and aniline, was demonstrated for the ®rst time
as a novel approach for designing such remediation
processes. 13C-NMR spectroscopy was used to identify
the Meisenheimer complex formed between TNT and
cysteine or aniline. Hydride±Meisenheimer complexes
have been clearly identi®ed as playing a key role in the
biodegradation of nitroaromatic compounds (Spain,
1995; Haidour and Ramos, 1996; Rieger et al., 1999).
In the past decades, nitroaromatics have been widely
disposed to soil and groundwater as dyes, pesticides,
and explosives (Hartter, 1985). Among nitroaromatic
compounds, 2,4,6-trinitrotoluene (TNT) has been
known as one of the widespread, mutagenic, and recal-
citrant pollutants (Won et al., 1976; Walker and
Kaplan, 1992; Rieger and Knackmuss, 1995). Many
years after the production activities were stopped, TNT
concentrations of 80 mg/l and 500 mg/l have been found
in process and groundwaters, respectively (Hao et al.,
1993; Price et al., 1997). These sites need to be reme-
diated as the potential hazard of TNT to human health
as well as its toxicity for higher and lower organisms have
been demonstrated. Several treatment techniques for
TNT-contaminated water are known such as biotreat-
ment, carbon adsorption, incineration, wet air oxidation
and chemical transformation (Spain, 1995; Hundal et
al., 1997; Held et al., 1997). Binding processes constitute
a potential to initiate soil clean-up (Verstraete and
2. Materials and methods
2.1. Treatment of water polluted with TNT by addition
of several amino compounds
A mixture of 1/1 (by vol.) H2O/d6-acetone with a
concentration of 0.2 mM TNT (Sigma Aldrich, Bel-
gium) was used for initial tests. The remaining amount
of TNT was measured 2 h after addition of 5.5 mM
cysteine (VEL, Belgium), 5.5 mM Fe2+/Fe3+ (VEL,
Belgium) and a combination of both.
* Corresponding author. Tel.: +32-9-264-59-76; fax: +32-9-264-
62-48.
E-mail address: willy.verstraete@rug.ac.be (W. Verstraete).
For subsequent tests, several l-amino acids (arginine,
methionine, serine, tyrosine, lysine, glycine, cysteine;
0269-7491/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0269-7491(00)00077-4

504
F. Fant et al. / Environmental Pollution 111 (2001) 503±507
VEL, Belgium) were added at a ®nal concentration of
0.9 mM. The solution contained 0.3 mM TNT.
To investigate the role of the amino group, equimolar
quantities of aniline, allylthiourea, diethylamine and
diphenylamine were added to a solution containing 0.4
mM TNT. The residual amount of TNT was deter-
mined after one day±night cycle.
On a solution of 0.3 mM TNT, the concentration
dependency for cysteine and aniline was measured for
®nal amino-compound concentrations of 0.0, 0.3, 0.9,
and 3.0 mM. One-half of the solution was kept in the
dark and the other half was exposed to 10 day±night
cycles.
the addition of cysteine eliminated 96% of free TNT
while the addition of Fe(II) or Fe(III) to a TNT solution
caused 91±94% disappearance of TNT. Simultaneous
treatment with cysteine and iron had no additional
e€ect on the decrease of free TNT compared to the
treatment with cysteine alone.
In order to gain more insights on the reaction
mechanism, several amino acids were tested at a con-
centration of 0.9 mM. After one day±night cycle of incu-
bation, 68±100% of free TNT disappeared from the
solution (Table 1). Thus, it seems that di€erent types of
amino acids could decrease the amount of free TNT in
water. To identify which of the two functional groups of
the amino acid (the amino or acid group) is involved in
reducing free TNT, equimolar quantities of several
amines were added to the TNT solution. Among four
amino-compounds tested, only aniline caused a sig-
ni®cant decrease of TNT as 86% of free TNT was
eliminated (Table 1). On this basis it can be concluded
that the amino group is the functional group that binds
TNT. However, not only is this functional group
important but also the overall structure of the com-
pound. In the case of diphenylamine, no TNT dis-
appearance was observed. An explanation could be that
the formation of a Meisenheimer complex (see below) is
probably hampered due to sterical hindrance of the two
aromatic rings.
The amount of amino-compound necessary to get
100% TNT disappearance has been investigated and
Table 2 shows that there is a minimal concentration of 3
mM cysteine or 0.3 mM aniline needed to totally bind
0.3 mM TNT. These results suggested that aniline binds
TNT better than cysteine.
When a TNT solution was standing for some days in
daylight, a red coloration due to a photochemical reac-
tion (Schmelling et al., 1998) was observed. The in¯uence
of light on a TNT solution in the presence of cysteine or
2.2. Photospectrometry
TNT was quanti®ed by a photospectrometric method
described by Jenkins (1990). Fifteen millilitres of acetone
was added to 10 ml of the water sample. Then, one drop
of 10% tetrabutylammonium bu€er solution was added
and the red coloration measured after 15 min at 550 nm.
2.3. NMR spectroscopy
Due to the insensitivity of this technique, the samples
of 1/1 (by vol.) H2O/d6-acetone contained 4 mM TNT
and 12 mM cysteine, except for the equimolar solution
that contains 4 mM TNT and 4 mM cysteine. The same
equimolar concentrations were used for the aniline/
TNT solution (in acetone) and for TNT dissolved in
acetone and in 9/1 (by vol.) acetonitrile/d3-acetonitrile.
The chemical shift is referenced relative to the methyl of
acetone (at 29.2 ppm for 13C). Small amounts of chro-
mium acetylacetonate (Ventron Alfaproducts, USA)
were added to reduce the relaxation times in order to
observe all quaternary carbon resonance signals.
The 1D proton decoupled 13C-NMR spectra were
recorded at a 13C frequency of 90.6 MHz and at room
temperature. The spectral width was 21.7 kHz (70,000
scans) and no relaxation delay was used. The spectral
resolution of the 32 K spectra was 1.3 Hz/pt. Prior to
Fourier transformation, the data were multiplied with
an exponential window function (LB 3.0 Hz) to increase
the sensitivity.
Table 1
E€ect of addition of amino acids (0.9 mM) and amines (0.9 mM) on
the disappearance of free 2,4,6-trinitrotoluene (TNT) after 1 day of
incubation
Solution
TNT decrease (%)
0
TNT (0.3 mM)
TNT (0.3 mM)+arginine
TNT (0.3 mM)+methionine
TNT (0.3 mM)+serine
TNT (0.3 mM)+tyrosine
TNT (0.3 mM)+lysine
TNT (0.3 mM)+glycine
TNT (0.3 mM)+cysteine
68
84
3. Results and discussion
99
99
3.1. Treatment of water polluted with TNT by addition
of several amino-compounds
100
99
98
VanBeelen and Burris (1995) described a reduction of
free TNT in the presence of Fe2+ and cysteine provided
1 h of incubation. This experiment was repeated and the
results showed that iron is not required to decrease the
amount of free TNT when cysteine was added. Indeed
TNT (0.4 mM)+aniline
86
0
TNT (0.4 mM)+allylthiourea
TNT (0.4 mM)+diethylamine
TNT (0.4 mM)+diphenylamine
0
0

F. Fant et al. / Environmental Pollution 111 (2001) 503±507
505
Table 2
Table 3
13C chemical shifts (in ppm) of 2,4,6-trinitrotoluene (TNT), cysteine
and aniline for di€erent samples at room temperature
Decrease in free 2,4,6-trinitrotoluene (TNT) (0.3 mM) for di€erent
concentrations of cysteine and aniline, incubated in daylight and in
darkness, after one and 10 day±night cycles
Chemical shift (ppm)
Solution
Decrease (%)
After 1 day
TNTa
TNTb
TNT/
Cysc
TNT/
Cysd
Anilinee
TNT/
anilinef
After 10 days
TNT
Daylight
Darkness
Daylight
Darkness
Daylight
Darkness
Daylight
Darkness
Daylight
Darkness
Daylight
Darkness
Daylight
Darkness
12
0
45
±
TNT
C1
134.5
151.9
123.1
146.6
15.4
98.6
150.9
122.5
145.3
14.9
72.8
150.9
122.5
145.3
14.9
48.5
150.9
122.5
145.3
14.9
±
±
±
±
±
48.8
151.6
122.4
146.3
14.9
TNT+0.3 mM cysteine
TNT+0.9 mM cysteine
TNT+3 mM cysteine
TNT+0.3 mM aniline
TNT+0.9 mM aniline
TNT+3 mM aniline
95
81
67
C2,6
C3,5
C4
2.2
82
100
100
100
100
98
33
C7
100
100
100
100
100
100
100
100
Cys
CO
Cafree
±
±
±
±
±
±
±
±
±
±
171.1
64.3
55.9
33.1
24.9
171.1
±
±
±
±
±
±
±
±
±
±
±
100
100
100
100
100
Cacomplex
55.9
±
24.9
Cbfree
Cbcomplex
Aniline
C1
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
147.9
116.3
130.0
119.2
125.9
94.1
C2,6
C3,5
C4
129.7
120.3
aniline was tested. The results showed that the in¯uence
of light on the binding reaction was only detected when
the concentrations of cysteine and aniline were lower
than the minimal amount needed for 100% binding
(Table 2).
a
4 mM TNT solution in 9/1 (by vol.) acetontrile/d3-acetonitrile.
4 mM TNT solution in 1/1 (by vol.) H2O/d6-acetone.
b
c
4 mM TNT and 12 mM cysteine in 1/1 (by vol.) H2O/d6-acetone.
4 mM TNT and 4 mM cysteine in 1/1 (by vol.) H2O/d6-acetone
after one day±night cycle incubation.
d
3.2. Determination of the Meisenheimer complex between
TNT and cysteine or aniline by 13C-NMR spectroscopy
e
Previously reported chemical shift values for aniline (11).
4 mM TNT and 4 mM aniline in 1/1 (by vol.) H2O/d6-acetone.
f
Anionic s-adducts, also termed Meisenheimer com-
plexes, arise from covalent bond formation between
nucleophiles and electron-de®cient nitroaromatic com-
pounds (Buncel et al., 1995). Meisenheimer complexes
were ®rst identi®ed by 13C-NMR by Olah and Mayr
(1976). The 13C chemical shifts of the carbons in the
ring are the best parameters to locate the exact binding
position, because there is a linear relation between the
13C chemical shift and the charge distribution in the ring
(Olah and Mayr, 1976).
A 1D 13C spectrum of a TNT solution in acetonitrile
was used to assign all the chemical shifts of TNT (Table
3). In the 1D 13C spectrum of the TNT sample in water/
d6-acetone (Fig. 1a), only the chemical shift of C1 does
not agree with the chemical shifts measured in the ace-
tonitrile solution of TNT. This chemical shift has an
up®eld shift of 34.6 ppm which is consistent with the
fact that acetone also forms a Meisenheimer complex
with TNT (Foster and Fyfe, 1966). This was con®rmed
by an up®eld shift of 3.0 ppm of the methyl carbon
atoms of acetone, and the fact that the TNT solution
also has a red colour.
distinguished based on the di€erence in the intensity of
their signals. The 13C signals of cysteine are shifted
up®eld by approximately 10 ppm after complexation
with TNT. All carbon signals of cysteine are doubled,
with the exception of the carbonyl carbon. Comparison
of the 13C resonance signals of TNT before and after
addition of cysteine (Table 3) revealed that by adding
cysteine the signal of C1 moved from 98.6 to 72.8 ppm
(Fig. 1). After incubation of the equimolar sample during
a day±night cycle, the signal of C1 at 72.8 ppm moved to
aliphatic region (ꢀ=48.5 ppm) which is characteristic for
a sp3 carbon (Fig. 1).
Analysis of the 1D 13C-NMR spectra of the equimo-
lar aniline/TNT mixture (Table 3) showed that the che-
mical shift of the carbon atoms closest to the amino
group are shifted up®eld. This could be due to the
proximity of the aromatic ring of TNT. The change in
hybridisation of the C1 atom of TNT after complex
formation results in an up®eld shift of 85.7 ppm of the
chemical shift of this atom (Table 3).
Using NMR spectroscopy, it is thus shown that TNT
forms a Meisenheimer complex with cysteine and ani-
line in 1/1 (by vol.) H2O/d6-acetone (Scheme 1). The
chemical shift of C1 of TNT is by far the largest up®eld
shift (from 50 up to 90 ppm) since the hybridisation of
In the 1D 13C spectrum of a mixture of 4 mM TNT
and 12 mM cysteine, two sets of signals appeared for
cysteine, one from uncomplexed (free) and the other
from complexed cysteine (Table 3). Both forms could be

506
F. Fant et al. / Environmental Pollution 111 (2001) 503±507
Fig. 1. 13C-NMR spectrum of 2,4,6-trinitrotoluene (TNT) (4 mM) (a) without and (b) with cysteine (12 mM) in 1/1 (by vol.) H2O/d6-acetone at
room temperature. The resonance signals of chromium acetylacetonate are indicated by an asterisk.
this atom changes from sp2 to sp3 during formation of
the Meisenheimer complex (Buncel et al., 1995). If the
covalent bond is formed with C3 of TNT or if the anilide
from the aniline is formed, the observed chemical shifts
will be totally di€erent. Thus, this complex is pre-
ferentially formed on the C1 position but due to the pre-
sence of some low abundance impurities it cannot be ruled
out that a Meisenheimer complex is formed at other posi-
tions. Alternatively, these impurities could be a result of
disulphide bond formation by the cysteine or solvent
impurities. Under biological conditions it has been shown
Scheme 1. Schematic representation of the formation of a Meisenhei-
mer complex between 2,4,6-trinitrotoluene (TNT) and amino com-
pounds (R-NH2).

F. Fant et al. / Environmental Pollution 111 (2001) 503±507
507
that some bacteria can reduce the aromatic ring of dinitro-
and trinitro-compounds by the addition of a hydride ion
to form a hydride±Meisenheimer complex, which subse-
quently re-aromatizes with the elimination of nitrite
(Spain, 1995; Haidour and Ramos, 1996). Recently, Rie-
ger et al. (1999) reported the evidence for the formation of
a hydride±Meisenheimer complex of picric acid (2,4,6-tri-
nitrophenol) and its protonated form under physiological
conditions using Rhodococcus erythropolis HLPM-1. It
was also proved that these complexes are key inter-
mediates of denitration and productive microbial degra-
dation of picric acid (Rieger et al., 1999).
Haidour, A., Ramos, J.L., 1996. Identi®cation of products resulting
from the biological reduction of 2,4,6-trinitrotoluene, 2,4-dini-
trotoluene, and 2,6-dinitrotoluene by Pseudomonas sp. Environ-
mental Science and Technology 30, 2365±2370.
Hao, O.J., Phull, K.K., Davis, A.P., Chen, J.M., Maloney, S.W., 1993.
Wet air oxidation of trinitrotoluene manufacturing red water. Water
Environmental Research 65, 213±220.
Hartter, D.R., 1985. The use and importance of nitroaromatic chemi-
cals in the chemical industry. In: Rickert, D.E. (Ed.), Toxicity of
Nitroaromatic Compounds. Chemical Industry Institute of Tox-
icology Series. Hemisphere Publishing, New York, pp. 1±13.
Held, T., Draude, G., Schmidt, F.R.J., Brokamp, A., Reis, K.H.,
1997. Enhanced humi®cation as an in-situ bioremediation technique
for 2,4,6-trinitrotoluene (TNT) contaminated soils. Environmental
Technology 18, 479±487.
Hundal, L.S., Shea, P.J., Comfort, S.D., Powers, W.L., Singh, J.,
1997. Long-term TNT sorption and bound residue formation in
soil. Journal of Environmental Quality 26, 896±904.
4. Conclusions
Jenkins, T.F., 1990. U.S. Army Cold Regions Research and Engi-
neering Laboratory, Special report, pp. 90±98.
This work showed that in a H2O/d6-acetone mixture,
TNT forms a Meisenheimer complex with cysteine and
aniline. When an overdose of cysteine is present, the
equilibrium moves entirely to the Meisenheimer com-
plex. After exposure to daylight, the formation of the
complex is more enhanced. The addition of cysteine,
aniline or crude protein extracts to water and soils pol-
luted with TNT or any other nitroaromatic compound
could be used as a simple and rapid process for binding
the pollutant and initiating further biological remediation
(Verstraete and Devlieghere, 1997).
Olah, G.A., Mayr, H.J., 1976. Carbone-13 nuclear magnetic resonance
study of Meisenheimer complexes and their charge distribution
pattern. Organic Chemistry 41, 3448±3451.
Price, C.B., Brannon, J.M., Hayes, C.A., 1997. E€ect of redox poten-
tial and pH on TNT transformation in soil±water slurries. Journal
of Environmental Engineering ASCE 123, 988±992.
Rieger, P.G., Knackmuss, H.-J., 1995. Basic knowledge and perspec-
tives on biodegradation of 2,4,6-trinitrotoluene and related
nitroaromatic compounds in contaminated soil. In: Spain (Ed.),
Biodegradation of Nitroaromatic Compounds. Plenum Press, New
York, pp. 1±18.
Rieger, P.G., Sinnwell, V., Preuû, A, Francke, W., Knackmuss, H.-J.,
1999. Hydride±Meisenheimer complex formation and protonation
as key reactions of 2,4,6-trinitrophenol biodegradation by Rhodo-
coccus erythropolis. Journal of Bacteriology 181, 1189±1195.
Schmelling, D.C., Gray, K.A., Kamat, P.V., 1998. Radiation-induced
reactions of 2,4,6-trinitrotoluene in aqueous solution. Environ-
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Acknowledgements
Financial support was received from SOILS ltd. and
VLAR ltd. (Belgium). We thank B. Meyns for his kind
advice.
Spain, J.C., 1995. Biodegradation of nitroaromatic compounds.
Annual Review of Microbiology 49, 523±555.
VanBeelen, P., Burris, D.R., 1995. Reduction of the explosive 2,4,6-
trinitrotoluene by enzymes from aquatic sediments. Environmental
Toxicology and Chemistry 14, 2115±2123.
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