Applied Magnetic Resonance

, Volume 47, Issue 6, pp 627–641 | Cite as

Kinetics of Rapid Covalent Bond Formation of Aniline with Humic Acid: ESR Investigations with Nitroxide Spin Labels

  • Michael MatthiesEmail author
  • Kevin Glinka
  • Marius Theiling
  • Kalman Hideg
  • Heinz-Jürgen Steinhoff


The bioavailability of many soil contaminants depends on their interaction with the soil organic matter. The paper presents a new approach of using stable paramagnetic spin labels for investigating the kinetics of covalent binding of specific xenobiotic functional groups with humic acids, a major organic matter fraction. Leonardite humic acid (LHA) was incubated with the nitroxide spin labels amino-TEMPO (4-amino-2,2,6,6-Tetramethylpiperidin-1-oxyl) and anilino-NO (2,5,5-Trimethyl-2-(3-aminophenyl)pyrrolidin-1-oxyl), respectively, which contain an aliphatic or aromatic functionality susceptible to interaction with LHA. Electron spin resonance spectra of LHA samples without and with the enzyme laccase were recorded at X-band frequency (9.43 GHz) at room temperature and neutral pH. Binding was detected by a pronounced broadening of the spectral lines after incubation of LHA for both spin labels. The development of a broad signal component in the spectrum of anilino-NO indicated the immobilization due to strong binding of the aniline group. The reorientational correlation time of bound anilino-NO is more than two orders of magnitude greater than that of the free label. The ratio of the amount of bound to the unbound species was used to determine the kinetics of the covalent bond formation. Reaction rate constants of 0.16 and 0.01 min−1 were determined corresponding to half-times of 4.3 and 69.3 min, respectively. Treatment of LHA with laccase enhanced the amount of the reacting anilino-NO species by a factor of 7.6, but left the reaction rate unaltered. Oxidative radical coupling was excluded using the spin trap agent n-tert-butyl-alpha-phenylnitrone.


Electron Spin Resonance Aniline Humic Substance Humic Acid Electron Spin Resonance Spectrum 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We kindly acknowledge the lab assistance of Elena Bondarenko. Support of the Hungarian National Research Fund OTKA K104956 is greatly acknowledged.

Compliance with Ethical Standards

Conflict of interest

The authors declare no competing financial interest.


  1. 1.
    M. Kästner, K.M. Nowak, A. Miltner, S. Trapp, A. Schäffer, Crit. Rev. Environ. Sci. Technol. 44, 2107 (2013)CrossRefGoogle Scholar
  2. 2.
    N. Senesi, Sci. Total Environ. 123/124, 63 (1992)CrossRefGoogle Scholar
  3. 3.
    F. Führ, H. Ophoff, Pesticide Residues in Soil (Wiley, Weinheim, 1998)Google Scholar
  4. 4.
    B. Gevao, K.T. Semple, K.C. Jones, Environ. Pollut. 108, 3 (2000)CrossRefGoogle Scholar
  5. 5.
    E. Barriuso, P. Benoit, I.G. Dubus, Environ. Sci. Technol. 42, 1845 (2008)ADSCrossRefGoogle Scholar
  6. 6.
    G.E. Parris, Environ. Sci. Technol. 14, 1099 (1980)ADSCrossRefGoogle Scholar
  7. 7.
    J.-M. Bollag, C. Myers, Sci. Total Environ. 117–118, 357 (1992)CrossRefGoogle Scholar
  8. 8.
    E.J. Weber, D. Colón, G.L. Baughman, Environ. Sci. Technol. 35, 2470 (2001)ADSCrossRefGoogle Scholar
  9. 9.
    C. Achtnicht, E. Fernandes, J.-M. Bollag, H.-J. Knackmuss, H. Lenke, Environ. Sci. Technol. 33, 4448 (1999)ADSCrossRefGoogle Scholar
  10. 10.
    G. Dawel, M. Kästner, J. Michels, W. Poppitz, W. Günther, W. Fritsche, Appl. Environ. Microbiol. 63, 2560 (1997)Google Scholar
  11. 11.
    K.A. Thorn, K.R. Kennedy, Environ. Sci. Technol. 36, 3787 (2002)ADSCrossRefGoogle Scholar
  12. 12.
    E.J. Weber, D.L. Spidle, K.A. Thorn, Environ. Sci. Technol. 30, 2755 (1996)ADSCrossRefGoogle Scholar
  13. 13.
    K.A. Thorn, P.J. Pettigrew, W.S. Goldenberg, Environ. Sci. Technol. 30, 2764 (1996)ADSCrossRefGoogle Scholar
  14. 14.
    H. Li, L.S. Lee, Environ. Sci. Technol. 33, 1864 (1999)ADSCrossRefGoogle Scholar
  15. 15.
    D. Colón, E.J. Weber, G.L. Baughman, Environ. Sci. Technol. 36, 2443 (2002)ADSCrossRefGoogle Scholar
  16. 16.
    J.-M. Bollaq, Environ. Sci. Technol. 26, 1876 (1992)ADSCrossRefGoogle Scholar
  17. 17.
    T. Müller, I. Rosendahl, A. Focks, J. Siemens, J. Klasmeier, M. Matthies, Environ. Pollut. 172, 180 (2013)CrossRefGoogle Scholar
  18. 18.
    M. Förster, V. Laabs, M. Lamshöft, J. Groeneweg, M. Krauss, M. Kaupenjohann, W. Amelung, Environ. Sci. Technol. 43, 1824 (2009)ADSCrossRefGoogle Scholar
  19. 19.
    A. Gulkowska, B. Thalmann, J. Hollender, M. Krauss, Chemosphere 107, 366 (2014)CrossRefGoogle Scholar
  20. 20.
    H.M. Bialk, A.J. Simpson, J.A. Pedersen, Environ. Sci. Technol. 39, 4463 (2005)ADSCrossRefGoogle Scholar
  21. 21.
    H.M. Bialk, C. Hedman, A. Castillo, J.A. Pedersen, Environ. Sci. Technol. 15, 3593 (2007)ADSCrossRefGoogle Scholar
  22. 22.
    H.M. Bialk, J.A. Pedersen, Environ. Sci. Technol. 42, 106 (2008)ADSCrossRefGoogle Scholar
  23. 23.
    A. Gulkowska, M. Sander, J. Hollender, M. Krauss, Environ. Sci. Technol. 47, 2102 (2013)Google Scholar
  24. 24.
    J.P. Klare, H.-J. Steinhoff, Spin Labeling EPR. Photosynth. Res. 102, 377 (2009)CrossRefGoogle Scholar
  25. 25.
    J.P. Klare, H.J. Steinhoff, in Book Series: Structure and Bonding, vol. 152, ed. by C.R. Timmel, J.R. Harmer (Springer, Berlin, 2013), pp. 205–248Google Scholar
  26. 26.
    A.D. Steen, C. Arnosti, L. Ness, N.V. Blough, Mar. Chem. 101, 266 (2006)CrossRefGoogle Scholar
  27. 27.
    C. Lattao, X. Cao, Y. Li, J. Mao, K. Schmidt-Rohr, M.A. Chapell, L.F. Miller, A.L. dela Cruz, J.J. Pignatello, Environ. Sci. Technol. 46, 12814 (2012)ADSCrossRefGoogle Scholar
  28. 28.
    P. Franchi, M. Lucarini, P. Pedrielli, G.F. Pedulli, Chem. Phys. Chem. 3, 789 (2002)Google Scholar
  29. 29.
    G.R. Buettner, Free Radic. Biol. Med. 3, 259 (1987)CrossRefGoogle Scholar
  30. 30.
    S. Gadányi, T. Kálai, J. Jekö, Z. Berente, K. Hideg, Synthesis 32, 2039 (2000)CrossRefGoogle Scholar
  31. 31.
    K. Stolze, N. Udilova, H. Nohl, Acta Biochim. Polon. 47, 923 (2000)Google Scholar
  32. 32.
    J. Fuchs, W.H. Nitschmann, L. Packer, O.H. Hankovszky, Free Rad. Res. Commun. 10, 315 (1990)CrossRefGoogle Scholar
  33. 33.
    A.P. Todd, R.J. Mehlhorn, R.I. Macey, J. Membr. Biol. 109, 41 (1989)CrossRefGoogle Scholar
  34. 34.
    Estimation Programs Interface Suite™ for Microsoft® Windows, v 4.11 (United States Environmental Protection Agency, Washington, 2012).
  35. 35.
    SPARC on‐line calculator (2015).
  36. 36.
    L.J. Berliner (ed.), Spin Labeling: Theory and Applications (Academic Press, New York, 1976)Google Scholar
  37. 37.
    M. Kawahigashi, H. Sumida, K. Yamamoto, J. Colloid Interface Sci. 284, 463 (2005)CrossRefGoogle Scholar
  38. 38.
    S.T.J. Droge, K.-U. Goss, Environ. Sci. Technol. 47, 798 (2013)ADSCrossRefGoogle Scholar
  39. 39.
    L. Urban, H.-J. Steinhoff, Mol. Phys. 111, 2873 (2013)ADSCrossRefGoogle Scholar
  40. 40.
    OECD Guideline for the Testing of Chemicals no. 308, Aerobic and Anaerobic Transformation in Aquatic Sediment Systems (Organisation of Economic Cooperation and Development, Paris, 2002)Google Scholar
  41. 41.
    A. Jezierski, F. Czechowski, M. Jerzykiewicz, Y. Chen, J. Drozd, Spectrochim. Acta A 56, 379 (2000)ADSCrossRefGoogle Scholar
  42. 42.
    M. Matthies, M. Theiling, K. Hideg, H.-J. Steinhoff, SETAC Europe Conference, Barcelona (2015), p. 27Google Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  • Michael Matthies
    • 1
    Email author
  • Kevin Glinka
    • 2
  • Marius Theiling
    • 2
  • Kalman Hideg
    • 3
  • Heinz-Jürgen Steinhoff
    • 2
  1. 1.Institute of Environmental Systems Research (USF), University of OsnabrückOsnabrückGermany
  2. 2.Physics DepartmentUniversity of OsnabrückOsnabrückGermany
  3. 3.Department of Organic and Medicinal ChemistryUniversity of PécsPécsHungary

Personalised recommendations