The Use of Infrared Microspectroscopy to Determine the Biotransformation of Benzotriazole by (Helianthus annuus L.) Sunflowers

1
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

2
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.
3
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

4
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.
5
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
6
Sunflowers grown in nutrient solutions containing
various concentrations of Benzotriazole (BT) for
14 days
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INTERNAL REFLECTING OBJECTIVE
IlluminatIR mounted between the body and the
eyepiece of an infinity corrected microscope
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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.
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Diamond ATR Spectra of xylem tissue of treated
and untreated dried secondary roots
745 cm-1 Aromatic CH bend
Relative
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Infrared Microspectroscopy at the Center for
Synchrotron Biosciences, Brookhaven National
Labs, Beamline U2B
Nicolet Nic Plan IR Microscope
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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

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IR spectra of root xylem of untreated and treated
Sunflower using a 100?m diameter aperture
1635 cm-1 aromatic stretch
1250 cm-1 CO stretch of lignin aromatic rings
745 cm-1 Aromatic C-H out-of-plane bending
1735 cm-1 CO stretch
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Assignment of Important Spectral Bands for Lignin
and Benzotriazole
Frequency Assignment Comments 745
cm-1 Aromatic C-H Benzene
ring in Benzotriazole

out-of-plane bending 1250 cm-1 Guaiacyl
ring breathing Guaiacyl syringyl subunits

with CO stretching 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
14
Untreated Sunflower Control
1750 cm-1
1250 cm-1
4 ?m section of secondary root from untreated
sunflower controls
745 cm-1
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Sunflowers treated with 20 mg/L Benzotriazole
1750 cm-1
1250 cm-1
4 ?m section of secondary root from sunflowers
treated with 20 mg/L benzotriazole
745 cm-1
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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

17
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.