The Use of Infrared Microspectroscopy to
Determine the Biotransformation of
Benzotriazole by (Helianthus annuus L.)
Sunflowers
Kenneth M. Dokken1, Lawrence C. Davis1, Larry E. Erickson2,
David L. Wetzel3, Nebojsa Marinkovic4, John A. Reffner5
1Department
of Biochemistry, Kansas State University
2Department of Chemical Engineering, Kansas State University
3 Microbeam Molecular Spectroscopy Laboratory, Kansas State University
4Center for Synchrotron Biosciences, Albert Einstein College of Medicine
5SensIR Technologies, Danbury, CT
ABSTRACT
Infrared microspectroscopy has been extensively applied in plant cell wall analysis to monitor
developmental changes. However, this technique is not widely used to study changes of plant
structures induced by exposure to organic contaminants. Previous studies of finely ground
secondary roots treated with various concentrations of benzotriazole used infrared
spectroscopy of KBr pellets to examine absorptions in the region of 749-775 cm-1. Increased
peak heights at 749 cm-1 were proportional to the concentration of benzotriazole to which the
plant had been treated. Peaks from lignin near 870 cm-1 decreased simultaneously. In this
study, sunflower plants were grown hydroponically in the presence of benzotriazole at
concentrations less than 1mM. At low concentrations, treated sunflower plants develop nearly
as well as untreated plants which allows optimal uptake and incorporation of benzotriazole
into the plant. Changes in the secondary structure due to uptake, incorporation, and/or
transformation of benzotriazole were monitored using diamond attenuated total reflectance
(ATR) and reflection absorption microspectroscopy. Diamond ATR spectra displayed the
same changes in spectra of secondary roots as seen in the traditional IR spectroscopy method
of KBr pellets. False color intensity maps of treated secondary root sections were produced
using absorption reflection infrared microspectroscopy at Brookhaven National Laboratories.
These showed changes in plant structure as well as the presence of aromatic CH peaks due to
incorporation of benzotriazole within the plant. The technique of IR microspectroscopy may
be used as a tool in phytoremediation to study changes of plant structure induced by the
presence of organic contaminants in soil and water.
Objectives
• Pinpoint the tissue location where benzotriazole is
being concentrated and/or transformed
• Determine the changes in plant structure induced
by exposure to benzotriazole
• Determine how the benzotriazole is being
transformed and/or incorporated into the plant
• To use this system as a model for determining the
fate of organic compounds in all types of
vegetation
Rationale
It is vital to understand the fate of organic contaminants within the plants
used for waste cleanup (phytoremediation). It is well documented that
many plants can biotransform organic contaminants. Major issues in the
field are whether transformed contaminants are still bioavailable and
whether they may have greater toxicity than the parent compound. As
currently understood, plant detoxification of xenobiotics typically depends
on oxidation, conjugation and sequestration. For various isotopically
labeled compounds, a large portion usually ends up in the lignin fraction of
plant cells. We propose to use benzotriazole as a model compound for
which we can specifically map out the locations of sequestration, which
often is initially found in the vacuole and ultimately in lignin. We know
that benzotriazole is transformed upon entry to the plant. Our working
hypothesis is that the peroxidatic system responsible for lignification
ultimately incorporates benzotriazole into a polymeric material that is no
longer bioavailable.
BENZOTRIAZOLE
Commonly used as a corrosion inhibitor in antifreeze, airplane deicing
fluids, gasoline, oil, lubricants, and heat exchanger fluids
Toxic to aquatic organisms and bacteria, possibly carcinogenic, UV
resistant (high stability), complexes with metals
Has been detected in ground and surface waters and has no known
bacterial degradation pathway
H
N
N
N
F.W. 119.13
Sunflowers grown in nutrient solutions
containing various concentrations of
Benzotriazole (BT) for 14 days
Leaves of treated plants turn
dark green and become crinkled
control
10 mg/L 20 mg/L 30 mg/L
BT
BT
BT
Higher concentrations of
BT lead to the production
of small highly branched
roots
INTERNAL REFLECTING OBJECTIVE
IlluminatIR® mounted
between the body and
the eyepiece of an
infinity corrected
microscope
Diamond Attentuated Total Reflectance (ATR) Spectroscopy
The diamond internal reflection objective design features the capability of
visible light viewing of the specimen surface both in the survey mode and
during data collection. A lens (see figure) inside of the secondary mirror is
focused by adjusting its height independent of the reflecting surface
The large ZnSe internal reflecting element surface is protected by a small
diamond cap. The curved surface assures optical contact with the specimen.
Sample Preparation for Diamond ATR Spectroscopy
Dry treated and untreated secondary roots of sunflower plants were laterally
dissected and placed on reflective slides The instrument used to obtain the
diamond ATR spectral data was an IlluminatIR® (SensIR® Technologies,
Danbury, CT) with a diamond ATR objective. 8cm-1 resolution was used with
256 scans coadded.
Longitudinal
section of dry
secondary root
Diamond ATR Spectra of xylem tissue of treated
and untreated dried secondary roots
Untreated root
Roots treated w/
60 mg/L BT
Relative
745 cm-1
Aromatic CH
bend
Infrared Microspectroscopy at the Center for Synchrotron
Biosciences, Brookhaven National Labs, Beamline U2B
Nicolet Nic Plan IR Microscope
Advantages of a Synchrotron IR
Source
• Brightness is 1000 times greater than
conventional (globar) sources for mid-IR
• High spatial resolution and spectral
resolution
• Decreased source noise
• Able to determine changes in structure at a
cellular level
IR spectra of root xylem of untreated and treated Sunflower
using a 100m diameter aperture
1635 cm-1
aromatic
stretch
1735 cm-1
CO stretch
1250 cm-1 CO
stretch of lignin
aromatic rings
745 cm-1
Aromatic C-H
out-of-plane
bending
Assignment of Important Spectral Bands for Lignin
and Benzotriazole
Frequency
Assignment
Comments
745 cm-1
Aromatic C-H
out-of-plane bending
Benzene ring in Benzotriazole
1250 cm-1
Guaiacyl ring breathing
with CO stretching
Guaiacyl –syringyl subunits
in lignin
1635 cm-1
Aromatic stretch
Aromatic rings of lignin
1750 cm-1
CO stretch
Unconjugated ketone and
carboxyl groups commonly
found in lignin
Sarkanen and Ludwig 1971, Lin-Vien et al. 1991 , Mohan and Settu 1993
Untreated
Sunflower Control
1750 cm-1
Log
µm
Co ntro l S unfl owe r 01 : P o i nt 1 @ (-31 75, -29 53)
60
0 .5
40
20
0 .0
0
3 500
0
3 000
50
2 500
2 000
1 50 (c m -1 )
W ave num bers
P o s i ti on (m i c rom eters )
1 00
1 500
1 000
1 500
1 000
2 00
1250 cm-1
Log
µm
6 0 Co ntro l S unfl owe r 01 : P o i nt 1 @ (-31 75, -29 53)
04
.50
20
0 .0
0
3 500
0
50
3 000
1 00
2 500
2 000
1 50
W ave num bers (c m -1 )
2 00
P o s i ti on (m i c rom eters )
745 cm-1
µm
60
Control plants do not
contain these bands!!!!
40
20
0
0
50
1 00
1 50
P o s i ti on (m i c rom eters )
2 00
4 m section of secondary
root from untreated
sunflower controls
-0 .0
Log
µm
1750 cm-1
02.40
0
0 .2
3 500
3 000
2 500
2 000
s u nflo wer 20 mg/L B T tip : P oint 1 @ (14 716 , 57 66)
W ave num bers (c m-1 )
0
1 00
Sunflowers treated
1 500
000
with 120
mg/L
Benzotriazole
2 00
Po s iti on (mic rom eters )
-0 .0
1250 cm-1
3 500
3 000
2 500
2 000
1 500
1 000
µm
W ave num bers (c m-1 )
20
0
0
1 00
2 00
P o s iti on (mic rom eters )
µm
745 cm-1
20
0
0
1 00
2 00
Po s iti on (mic rom eters )
4 m section of
secondary root from
sunflowers treated
with 20 mg/L
benzotriazole
Conclusions
• Diamond ATR and IR Microspectroscopy can be used as a
technique to detect changes in plant structure due to
exposure to organic contaminants.
• Benzotriazole becomes transformed and incorporated into
the sunflower plant presumably through lignin
– Presence of 745 cm-1 due to C-H bending in Benzotriazole
– More prominent bands at 1250 cm-1 and 1750 cm-1 due to
increased lignin production
References
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Acknowledgements
The authors thank SensIR Technologies for the use of their instruments and facilities in Danbury, CT. This
project was supported by U.S. Environmental Protection Agency under assistance agreement R-825550 through
the Great Plains/Rocky Mountain Hazardous Substance Research Center, Kansas Agricultural Experiment
Station and the KSU Microbeam Molecular Spectroscopy Laboratory. The authors would also like to thank
Cindy Chard-Bergstrom and Deborah St. Cyr for their help with microtoming the root sections.