The effect of UV and visible light radiation on natural humic acid

Geoderma 126 (2005) 291 – 299 www.elsevier.com/locate/geoderma

The effect of UV and visible light radiation on natural humic acid EPR spectral and kinetic studies K. Polewskia,*, D. Syawin´skaa, J. Syawin´skia, A. Pawlakb a

Department of Physics, August Cieszkowski Agricultural University, ul. Wojska Polskiego 38/42, 60-637 Poznan´, Poland b Faculty of Biotechnology, Jagiellonian University, Krako´w, Poland Received 3 October 2003; received in revised form 30 August 2004; accepted 1 October 2004 Available online 5 November 2004

Abstract The effect of polychromatic UV and visible light on the radical properties of humic acid (HA) extracted from composted shells of walnut tree Juglans regia was investigated. The exposure of HA solutions 2 mg/ml in 0.01 M Na2CO3 at pH=10.8 to the light from a 150-W xenon lamp increases the signal amplitude of electron paramagnetic resonance (EPR). Using the set of cut-off filters at 390, 340, 280 and 200 nm, the efficiency of free radical formation was determined. It shows the maximum efficiency of radicals generation in the range 280–340 nm, which corresponds to the maximum in absorption spectra of quinones and naphthoquinones in the UV range. Generation of the free radicals during visible light irradiation is connected with photosensitized properties of HA. The single exponential decay of the EPR signal observed after irradiation, calculated g-value and spectral and kinetic studies of photoinduced EPR amplitude changes in aerated and deaerated solutions indicate that the reactive oxygen species (ROS) and excited molecules in the triplet state are very probably the factors leading to the formation of stable quinoid type of free radical in HA. Observed linear increase of free radicals generated during irradiation indicates on the great capacity of HA to efficiently stabilize free radicals inside its structure. D 2004 Elsevier B.V. All rights reserved. Keywords: Humic acid; EPR; Semiquinones; Free radicals; Photodegradation; Juglone; UV radiation

1. Introduction The knowledge of chemical and physical properties of humic substances (HS) is essential in under-

* Corresponding author. Tel./fax: +48 61 848 7495. E-mail address: [email protected] (K. Polewski). 0016-7061/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.geoderma.2004.10.001

standing their interactions with environmental factors, particularly with the UV and visible light and oxygen. These interactions are photophysico-chemical in nature and depend on the chemical composition and structure of HS. The behavior of bulk humic substances (HS) or humic acid (HA) in reactions with light, oxygen and prooxidants present in soil and aquatic environment (Slawin´ska et al., 1975a,b; Slawin´ska and Michalska, 1978; Hoigne et al.,

292

K. Polewski et al. / Geoderma 126 (2005) 291–299

1989; Senesi, 1990; Senesi and Miano, 1994; Drozd et al., 1997; Chen et al., 2003) may differ significantly from those of individual HA component. In this respect, a high content of unpaired spins is an important factor as the organic matter of geoderma is far from thermal equilibrium with atmospheric oxygen. The main factor which slows down an unavoidable oxidation reaction of organic matter is the different multiplicity of the reactions substrates, i.e. the singlet of organic matter and triplet of molecular oxygen. The absorption of light by chromophores existing in humic acid (HA) may lead to alterations of HA structure and composition which is reflected by the changes in absorption, fluorescence, chemiluminescence, NMR, IR and EPR spectra (Senesi and Miano, 1994; Slawin´ski et al., 1998; Jezierski et al., 2000; Kalbitz et al., 2000; Piana and Zahir, 2000; Tratnyek et al., 2001; Imai et al., 2002; Chen et al., 2003). When quinone moieties in HS undergo one-electron reduction, free radicals of semiquinones and charge-transfer complexes are formed. They are efficient absorbers of light in a broad spectral range. It was shown that quinone moieties in HA are relevant not only to radical content but also to HA’s antioxidant activity (Polewski et al., 2002), regulation of redox reactions in metabolic plant and microbial processes (Kalyanaraman, 1990; Jurcsic, 1994; Scott et al., 1998) and metal-ion pollutants (Jezierski et al., 2000, 2002). Recently, a HA fraction from composted green shells of walnut tree (Juglans regia L.), containing a large amount of naphthoquinones, was obtained (Slawin´ska et al., 2002, 2004). Naphthoquinones, especially 5-hydroxy 1:4 naphthoquinone (juglone) were found in J. regia L and Juglans nigra (Blumenthal, 1998). Juglone and its derivatives occur in the roots, fruits, leaves and wood of J. regia L and exhibit a wide spectrum of pharmacodynamic activity, especially cytotoxicity and genotoxicity (Thompson, 1987; Babich and Stern, 1993). There is a large number of plants, soil microorganisms and animals sensitive to allelopathic polyphenolic and quinoid compounds like juglone that can be incorporated into HA during the humification process. However, no systematic studies have been done on these aspects of HA originating from humified litter of Juglans species.

For all these reasons, it is interesting to examine the photophysico-chemical and paramagnetic properties of HA obtained from composted plant residue abundant in hydroxynaphthoquinones. The aim of this work is to investigate the influence of UV and visible light on the natural humic acid from walnut shells which was isolated and characterized for the first time (Syawin´ska et al., 2002). The effect of the UV and visible radiation is investigated using EPR spectroscopy method. The applied cut-off filter limits, at 280 and 340 nm, correspond to the upper limits of UV-C and UV-A solar radiation which increasingly penetrates the Earth biosphere.

2. Materials and methods The HA was isolated from the composted hulls of walnut J. regia rich in naphthoquinones. Details of the preparation procedure and physico-chemical properties of the HA are given elsewhere (Slawin´ska et al., 2002). EPR spectra of solid HA were measured in a 2-mm quartz tube. EPR measurements of aqueous samples in 0.01 M Na2CO3 at pH=10.8 of 2 mg/ml HA were carried out at room temperature using quartz flat cell and in situ irradiation of the samples (Rozanowska et al., 1995). Electron paramagnetic resonance (EPR) measurements of solid and aqueous samples of HA were carried out using a Bruker ESP 300 E spectrometer operating at X-band at 9.5-GHz frequency and equipped with 100-kHz field modulation, modulation amplitude 0.946 G, sweep width 110 G, conversion time 82 ms, time constant 327 ms. The spectrometer type SE/X 2544 (Radiopan, Poznan´, Poland) equipped with a resonance chamber RCX 660 was used to obtain spectra of solid HA sample and a standard. EPR signals were recorded at room temperature with the following settings: microwave power 3 mW, time constant 1 s, scan rate of 20 mT/2 min, modulation frequency of 100 kHz/0.2 mT and a microwave frequency of 9.45 GHz. The signals were used to calculate the peak-to-peak amplitude (a.u.). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) was employed as the standard to determine the g coefficient ( g=2.005) and concentration of spins. Care was taken to ensure constant conditions

K. Polewski et al. / Geoderma 126 (2005) 291–299

of humidity (24F2%) and temperature (22F1) 8C during sample preparation and the reproducibility of EPR measurements. The concentration of unpaired spins in HA (N p) was calculated from the formula n
o 2 Np ¼ Ns Ip DBp =Is ðDBs Þ2

ð1Þ

where N p and N s are the spin concentration in HA and in standard (DPPH), I p and I s are amplitudes of HA and standard and DB p and DB s are peak-to-peak line width of the EPR spectra, respectively. Samples were irradiated with polychromatic radiation 200–600 nm using a high-pressure xenon lamp (Photomax 150 W, Oriel) equipped with quartz lenses. Three long pass cut-off filters at 390, 340 and 280 nm were used to select different irradiation ranges. Irradiations were carried out at room temperature in aerated samples and in deaerated samples by purging the sample for 40 min with argon. The lamp output at the optical cell for specific wavelengths was monitored with an International Light IL1700 radiometer using calibration factors delivered by the manufacturer; values of energy flux incident on the sample are reported in units of W m2.

293

3. Results 3.1. EPR spectra of solid HA The EPR spectrum of the solid HA is presented in Fig. 1. Spin density is 4.41019 spin g1 and line width is 0.645 mT with g=2.0048. As Fig. 1 shows, the spectrum is broad without any fine structure which is very typical for the EPR spectra of other HA. In order to obtain information on mechanisms of the relaxation of absorbed microwave energy by HA, the progressive saturation experiments with increasing microwave power were performed. The empirical expression used to fit saturation data obtained for protein is given by Galli et al. (1996). Lately this method was used to calculate relaxation process in organic free radicals in peat humus samples (Novotny and Martin-Neto, 2002). The power saturation curve for the doubleintegrated intensity of the in-phase first harmonic absorption spectra is given as an inset in Fig. 1. From such experiment, we may calculate P 1/2 and from fitting procedure to Eq. (2), it is possible to obtain inhomogeneity parameter b. 
b=2 Y V ¼ KP1=2 1 þ P=P1=2 ð2Þ where YV is the EPR derivative signal amplitude, K is a constant, P is microwave power, P 1/2 is

Fig. 1. EPR spectrum of solid HA measured at 2-mW microwave power with g=2.0048. The inset shows the relation between first-derivative area versus increasing microwave power.

294

K. Polewski et al. / Geoderma 126 (2005) 291–299

the microwave power at half-saturation, and b is the inhomogeneity parameter. Applied least square fitting procedure to the data presented in Fig. 1 inset gave b value equal to 0.77 which suggests that high dipolar-relaxation enhancement occurs in the solid state phase of HA. 3.2. EPR spectra of aqueous samples The first-derivative EPR spectra of two dark and two irradiated solutions of HA (2 mg/ml) in 10 mM Na2CO3 in the presence of air are given in Fig. 2. The spectra are characterized by an intense symmetrical line without any hyperfine structure, which indicates the presence of complex organic free radicals in humic substances (Senesi, 1990). For all samples, the spectra are broad and structureless with the peak-to-peak width 0.559 mT which does not change during irradiation. The resonance line is characterized by a splitting factor ( g-value) for air samples ranging from 2.0044 for dark samples to 2.0048 for the irradiated samples. The deareated samples practically do not show any dependence on the number of dark/light cycles, the g-value is constant for dark and irradiated samples and equals 2.0048.

Fig. 2. First-derivative EPR spectra of aqueous 2 mg ml1 HA in 0.1 M Na2CO3 of: (a) dark and irradiated sample in the presence of air; (b) dark and irradiated in deaerated (Ar) samples. Samples are irradiated with kN200 nm. The spectra are taken at 9.5-GHz frequency with 100-kHz field modulation, modulation amplitude 0.946 G, sweep width 110 G, conversion time 82 ms, time constant 327 ms and 1 mW of microwave power.

In aerated samples, the increase of signal intensity during irradiation is higher than in deaerated sample, Fig. 2. However, the amplitude of the changes (the ratio of signal intensities of irradiated to dark sample) is significantly higher in the deaerated sample, (2.8) compared to the aerated sample, (1.9). These characteristics are consistent with the presence of local organic free radicals of semiquinone nature, conjugated with an aromatic system present in HA. An increased stabilization of substituted semiquinone radicals as semiquinone ions due to the high pH and the presence of oxygen in the sample may be expected (Senesi and Schnitzer, 1977). The samples irradiated using cut-off filters with increasing wavelengths, i.e. N280, N340 and N390 nm exhibit similar relations of the EPR spectra regarding dark/light ratio as described for the sample irradiated with radiation N200 nm discussed in the previous paragraph and shown in Fig. 2. However, the amplitude of the change depends on the spectral range used to irradiate the sample. The relations between the spectral ranges of the irradiation, the initial EPR signal amplitude change during irradiation and the optical absorption spectrum of HA are given in Fig. 3. The obtained absorption spectrum is typical for humic substances where absorbance monotonically increases with increasing energy of light. The positions of cut-off wavelengths of the used filters are presented on the abscissa as vertical lines where theirs height is related to the energy, given on the right axis, delivered to the sample. The relations between the initial EPR amplitude change and spectral irradiation range are given in the inset in Fig. 3. It shows that the irradiation with cut-off filter transmitting at kN390 nm increases the EPR signal amplitude. Irradiation with cut-off filter at kN340 nm increases the amplitude three times compared when irradiated with visible light. The irradiation with cut-off filters transmitting at wavelengths N200 and N280 nm increases the amplitude of the signal four times than that observed for the visible light (N390 nm). This increase is the same for the both filters. Taking into consideration increasing absorption coefficient and increasing irradiation energy, one may expect that the increase of the EPR amplitude signal when irradiated with kN200 nm should be the highest. However, as can be seen from the inset in Fig. 3, irradiation with wavelengths lower than 280 nm does

K. Polewski et al. / Geoderma 126 (2005) 291–299

295

Fig. 3. Absorption spectrum of 2 mg ml1 of HA in 0.1 M Na2CO3 in 0.3-mm cuvette before irradiation. Vertical lines on the abscissa give values of the short-wavelength cut-off limit of the used filters, whereas the height shows the amount of irradiation energy delivered to the sample at given irradiation range, right vertical scale. The inset shows the initial amplitude increase of EPR signal during irradiation with given filter in HA aerated sample. The amplitude of the unirradiated sample is taken as a zero level.

not generate a higher EPR signal despite the highest energy of irradiation and highest absorption of the sample in this range. This indicates that the main sources of the free radicals generated in HA during irradiation are the aromatic structures that absorb in the range above 280 nm. Irradiation with cut-off filter N340 nm gives the amplitude change about 80% of that observed at a 280-nm irradiation. This points to the structures like quinones, aromatic ketons and polyphenols, which are known to absorb in this range. The observed increase in EPR amplitude at 280-nm irradiation compared to that observed at 340-nm irradiation should be rather attributed to the increasing absorption coefficient of aromatic components than to existence of another specific component of HA. The changes in the amplitude of EPR signal observed for cut-off filter N390 nm are low despite the sample is irradiated with full range of visible light that gives a large number of absorbed photons compared to the UV ranges. Because the energy in this range is too low to initiate any direct photoionization process, then one may rather expect that observed changes are connected with photosensitized mechanism. Numerous studies show that humic substances act as photosensitizers and reaction substrates leading to the production of transient intermediates like reactive

oxygen species or formation of relatively stabile, photoinduced free radicals (Zepp et al., 1985; Haag and Hoigne, 1986; Frimmel and Christman, 1988; Blough and Zepp, 1995; Aguer et al., 2002). 3.3. Kinetics of EPR signal In order to elucidate the possible mechanisms of the interaction between UV radiation and HA, temporal changes in the EPR signal have been investigated. The kinetics of EPR amplitude signal during irradiation of aerated and deaerated samples were measured. The samples were irradiated with four spectral ranges, above 390, N340, N280 and N200 nm. Because for all used irradiation ranges, the kinetic traces are similar, except the magnitude of amplitude changes, we present results obtained for the HA sample irradiated with cut-off filter N280 nm. The results of change of EPR amplitude during irradiation are shown in Fig. 4. In the aerated sample, when irradiated, Fig. 4a, the amplitude first increases very sharply to reach the maximum and then slowly decays to 60% of its maximal value, area 2 and then start to increase, area 3. When the light is off, the signal slowly decays, area 4, to the starting dark level, area 1. Further irradiation of

296

K. Polewski et al. / Geoderma 126 (2005) 291–299

Fig. 4. The kinetics of the EPR amplitude change during irradiation with kN280 nm in the presence of air (a) 0 to 10 min; (b) 18 to 30 min and purged with argon (c) 0 to 10 min. Please take note the break in (b) on time axis between 10 and 18 min. Number in the circle refers to explanation given in text. The amplitude of the unirradiated sample is taken as a zero level.

the sample, Fig. 4b, causes increase of the signal amplitude to the value before shut-down and then constant linear increase, area 5. Turning the light off again, the signal decreases, area 6. We may notice that now the dark level is 20% higher compared to the starting dark level, area 1. This means during irradiation, stabile free radicals in HA sample have been generated. In the sample purged with argon, Fig. 4c, during irradiation the EPR amplitude sharply increases at the beginning and then slow, constant increase is observed, area 2. When the light is off, the signal slowly decays to dark level, area 3, which is now 40% higher compared to the initial level, before irradiation, area 3. Further irradiation of the sample returns the amplitude to the previous value before shut-off and linear constant increase continues, area 4. The signal decays observed after the light is off, areas 4 and 6 in Fig. 4a,b and area 3 in Fig. 4c are fitted with one component exponential decay (with the time constant 58 s and correlation coefficient 0.979) which describes the process of spin recombination. The observed difference in the kinetics of aerated and deaerated samples during first 4 min is connected with the presence of air. After the oxygen available in the aerated sample is exhausted in the reactions, the

sample kinetics appears to be very similar to the deaerated sample, see Fig. 4b and c. In deaerated samples during irradiation, a small but constant increase of EPR amplitude is observed. Our attempts to fit the amplitude increase during irradiation in deaerated samples with physically meaningful formulas has shown that this kinetic is best fitted by the superposition of the exponential increase at the very beginning and then linear increase. This linear response between the number of generated radicals and the irradiation indicates on the great capacity of HA to stabilize formed radicals in its structure.

4. Discussion Investigated natural HA is characterized by elemental analysis showing a high content of oxygen 47.6% and low of carbon 44.8% (Slawin´ska et al., 2002) which indicates that it may contain more phenolic and ketonic functional groups. A quantitative EPR analysis gives a high paramagnetic spin count. Reports (Steelink and Tollin, 1966; Slawin´ska et al., 1975a,b, 2002; Slawin´ski et al., 1978a,b; Senesi, 1990) have shown that the number of spins in humic

K. Polewski et al. / Geoderma 126 (2005) 291–299

substances ranges from 1016 to 1019 spins/g of HA. This number includes primary and secondary radicals that generate EPR signal. Another source of free radicals formation is autooxidation which may initiate the formation of secondary radicals. The assignment of the observed EPR signal is not an easy task because of the simultaneous existence of a several types of radicals, similar proton environment for different carbon atoms, acid–base equilibrium of radicals, dependence on the pH and temperature. Those factors cause that the observed EPR spectrum for the HA is broad and structureless. Steelink and Tollin (1966) suggested that simple quinone–phenol compounds could be characteristic of EPR properties of humic substances. This EPR signal is attributed to the semiquinone radical units possibly conjugated to aromatic rings, although contributions from methoxybenzene and nitrogen-associated radicals cannot be excluded (Senesi, 1990). It also depends on various laboratory conditions such as pH, irradiation, acidhydrolysis, methylation, and temperature (Senesi, 1990; Senesi and Miano, 1994). The microwave progressive power saturation method has been used to evaluate the electron spinrelaxation rate and to distinguish between exchange and dipolar contributions to spin-relaxation enhancement, a process that occurs when a slow relaxation spin is near a rapidly relaxing species (Galli et al., 1996). The obtained b value is less than 1 although not physically meaningful, bb1 can be utilized to estimate the magnitude of dipolar spin-relaxation enhancement. Low value of the inhomogeneity parameter b and high content of free radicals indicate the presence of strong dipole–dipole interaction between spins located on the HA aromatic subunits. The above-presented consideration indicates the significant dipolar interactions between adjacent paramagnetic centers of quinoid radical forms of HA components. During irradiation of quinones or phenols, only primary free radicals are generated by the photoionization and photohomolytic processes (Kalyanaraman, 1990). In case of phenols, they are formed by the abstraction of the hydrogen atom from hydroxy group at phenolic oxygen. Additionally, in the presence of oxygen, the formation of a superoxide radical is observed. It has been shown that humic substances act as sensitizers and precursors for the production of

297

reactive intermediates including singlet oxygen, superoxide anion or hydrogen peroxide (Slawin´ska et al., 1975a,b; Slawin´ski et al., 1978a,b; Cooper et al., 1989; Hoigne et al., 1989; Canonica and Hoigne, 1995; Aguer and Richard, 1996). From the kinetic and spectral studies on the aerated and deaerated samples presented in this work, it seems that several reactive oxygen species (ROS) are involved in the phototransformation of investigated natural HA. However, ROS are shortliving species and because we used a steady-state EPR, the observed signal must be due to relatively long-lived radicals. This suggests that observed free radical signal is connected with secondary radicals which are formed during oxidation reactions of HA components and ROS. It means that except peroxyl-type radicals, also reactive triplet states of aromatic subunits like carbonyl chromophores may be involved. Chemiluminescent studies (Slawin´ska and Michalska, 1978) and EPR (Canonica and Hoigne, 1995) regarding the formation of excited states during HA irradiation suggest that carbonyl-type chromophores could be responsible for the photoreactivity. The single exponential decay of EPR signal observed after irradiation implies the presence either of a single chromophore photobleaching with a single rate constant, or multiple chromophores decaying with identical rate constants. Calculated g-value indicates that under different irradiation ranges, one radical species formation occurs. The calculated maximum efficiency of radicals generation is observed for the light in the range 280–340 nm which corresponds to maximum absorption spectra in the UV range of quinones and naphtoquinones. Taking into consideration all above data and discussion, we suggests that quinoid moiety, with carbon-centered free radical, is responsible for the stabile EPR signal observed in HA during UV irradiation. The generation of the free radical signal during visible light irradiation of HA suggests that photosensitized mechanism is also involved in this process. A positive correlation between oxidation capacity and the stable free radical content of humic acid was reported (Struyk and Sposito, 2001), however, this latter property could account only for a less than few percent of electrons transferred between humic acid and I2 oxidant. The phenomena of linear EPR signal increase during irradiation and increasing level of bdarkQ signal after irradiation are strong indications that the investigated HA exhibits great capacity to

298

K. Polewski et al. / Geoderma 126 (2005) 291–299

efficiently stabilize free radicals generated during irradiation with UV and visible light.

Acknowledgment This work is supported by grant 3 P04G 016 22 from the State Committee for Scientific Research, Poland. Our special thanks to Prof. Boleslaw Gonet from Pommeranian Medical University in Szczecin for a kind help in EPR measurements and Mr. Piotr Rolewski for his technical assistance at humic acid samples preparation.

References Aguer, J.-P., Richard, C., 1996. Reactive species produced on irradiation at 365 nm of aqueous solutions of humic acids. J. Photochem. Photobiol., A Chem. 93, 193 – 198. Aguer, J.P., Richard, C., Trubetskaya, O., Trubetskoj, O., Leveque, J., Andreux, F., 2002. Photoinductive efficiency of soil extracted humic and fulvic acids. Chemosphere 49, 259 – 262. Babich, H., Stern, A., 1993. In vitro cytotoxicities of 1,4naphthoquinones and hydroxylated 1,4-naphthoquinones to replicating cells. J. Appl. Toxicol. 13, 353 – 358. Blough, N.V., Zepp, R.G., 1995. Reactive oxygen species in natural waters. In: Foote, C.S., Valentine, J.S., Greenberg, A., Liebman, J.F. (Eds.), Active Oxygen in Chemistry, vol. 2, Blackie Academic & Professional, pp. 280 – 333. Blumenthal, M., 1998. American Botanical Council. Wallnut null, Austin, TX, pp. 381 – 397. Canonica, S., Hoigne, J., 1995. Enhanced oxidation of methoxy phenols at micromolar concentration photosensitized by dissolved organic material. Chemosphere 30, 2365 – 2374. Chen, J., LeBoeuf, E.J., Dai, S., Gu, B., 2003. Fluorescence spectroscopic studies of natural organic matter fractions. Chemosphere 50, 639 – 647. Cooper, W.I., Zika, R.G., Petasne, R.G., Fischer, A.M., 1989. Sunlight-induced photochemistry of humic substances in natural waters: major reactive species. In: Suffet, I.H., MacCarthy, P. (Eds.), Aquatic Humic Substances: Influence on Fate and Treatment of Pollutants. Am. Chem. Soc., Washington, DC, pp. 333 – 362. Drozd, J., Gonet, S.S., Senesi, N., Weber, J. (Eds.), The Role of Humic Substances in the Ecosystems and in Environmental Protection. PTHS, Wrocyaw, Poland. Frimmel, F.H., Christman, R.F., 1988. Humic Substances and their Role in the Environment. John Wiley & Sons, Chichester, New York. Galli, C., Innes, J.B., Hirsh, D.J., Brudvig, G.W., 1996. Effects on dipole–dipole interactions on microwave progressive power saturation of radicals in proteins. J. Magn. Reson., Ser. B 110, 284 – 287.

Haag, W.R., Hoigne, J., 1986. Singlet oxygen in surface waters: 3. Photochemical formation and steady-state concentrations in various types of waters. Environ. Sci. Technol. 20, 341 – 348. Hoigne, J., Faust, B.C., Haag, W.R., Scully, F.E.J., Zepp, R.G., 1989. Aquatic humic substances as sources and sinks of photochemically produced transient reactants. In: Suffet, I.H., MacCarthy, P. (Eds.), Aquatic Humic Substances: Influence on Fate and Treatment of Pollutants. Am. Chem. Soc., Washington, DC, pp. 363 – 381. Imai, A., Fukushima, T., Matsushige, K., Kim, Y.H., Choi, K., 2002. Characterization of dissolved organic matter in effluents from wastewater treatment plants. Water Res. 36, 859 – 870. Jezierski, A., Czechowski, F., Jerzykiewicz, M., Chen, Y., Drozd, J., 2000. Electron paramagnetic resonance (EPR) studies on stable and transient radicals in humic acids from compost, soil, peat and brown coal. Spectrochim. Acta, Part A: Mol. Biomol. Spectrosc. 56A, 379 – 385. Jezierski, A., Czechowski, F., Jerzykiewicz, M., Golonka, I., Drozd, J., Bylinska, E., Chen, Y., Seaward, M.R., 2002. Quantitative EPR study on free radicals in the natural polyphenols interacting with metal ions and other environmental pollutants. Spectrochim. Acta, Part A: Mol. Biomol. Spectrosc. 58, 1293 – 1300. Jurcsic, I., 1994. Investigations of the mechanism of electron transmission and active oxygen generating humic acids supported by redox indicator. In: Senesi, N., Miano, T.M. (Eds.), Humic Substances in the Global Environment & Implications on Human Health. Elsevier, Amsterdam, pp. 311 – 316. Kalbitz, K., Geyer, S., Geyer, W., 2000. A comparative characterization of dissolved organic matter by means of original aqueous samples and isolated humic substances. Chemosphere 40, 1305 – 1312. Kalyanaraman, B., 1990. Characterization of semiquinone radicals in biological systems. Methods Enzymol. 186, 333 – 343. Novotny, M., Martin-Neto, L., 2002. Effects of humidity and metal ions on the free radicals analysis of peat humus. Geoderma 106, 305 – 317. Piana, M.J., Zahir, K.O., 2000. Investigation of metal ions binding of humic substances using fluorescence emission and synchronous-scan spectroscopy. J. Environ. Sci. Health, Part B, Pestic. Food Contam. Agric. Wastes 35, 87 – 102. Polewski, K., Syawin´ska, D., Syawin´ski, J., 2002. The role of ortoand para-semiquinones in the chemiluminescence and antioxidizing activity of humus substances. In: Stanley, P.E., Kricka, L.J. (Eds.), Bioluminescence & Chemiluminescence. World Scientific, New Jersey, pp. 161 – 164. Rozanowska, M., Ciszewska, J., Korytowski, W., Sarna, T., 1995. Rose-bengal-photosensitized formation of hydrogen peroxide and hydroxyl radicals. J. Photochem. Photobiol., B Biol. 29, 71 – 77. Scott, D.T., McKnight, D.M., Blunt-Harris, E.L., Kolesar, S.E., Lovley, D.R., 1998. Quinone moieties act as electron acceptors in the reduction of humic substances by humic-reducing microorganisms. Environ. Sci. Technol. 32, 2984 – 2989. Senesi, N., 1990. Application of ESR spectroscopy in soil chemistry. Stewart, B.A. Adv. Soil Sci. Springer Verlag, Heidelberg, pp. 77 – 130.

K. Polewski et al. / Geoderma 126 (2005) 291–299 Senesi, N., Miano, T.M., 1994. Humic Substances in the Global Environment and Implications on Human Health. Elsevier, Amsterdam. Senesi, N., Schnitzer, M., 1977. Effects of pH, reaction time, chemical reduction and irradiation on ESR spectra of fulvic acid. Soil Sci. 123, 224 – 234. Syawin´ska, D., Michalska, T., 1978. Singlet oxygen and chemiluminescence in the oxidation of polymers resembling humic acids. In: Ranby, J.F., Rabek, J. (Eds.), Singlet Oxygen. Reactions with Organic Compounds and Polymers. John Wiley & Sons, Chichester, New York, pp. 294 – 301. Syawin´ska, D., Syawin´ski, J., Sarna, T., 1975a. Effect of light on EPR spectra of humic acids. J. Soil Sci. 26, 93 – 99. Syawin´ska, D., Syawin´ski, J., Sarna, T., 1975b. Photoinduced luminescence and ESR signals of polyphenols and quinonepolymers. Photochem. Photobiol. 21, 393 – 396. Syawin´ski, J., Puzyna, W., Syawin´ska, D., 1978a. Chemiluminescence during photooxidation of melanins and soil humic acids arising from a singlet oxygen mechanism. Photochem. Photobiol. 28, 459 – 463. Syawin´ski, J., Puzyna, W., Syawin´ska, D., 1978b. Chemiluminescence in the photooxidation of humic acids. Photochem. Photobiol. 28, 75 – 81. Syawin´ski, J., Go´rski, H., Manikowski, H., 1998. EPR spectra of humic acids and melanins exposed to UV radiation and ozone. Curr. Top. Biophys. 23, 103 – 112.

299

Syawin´ska, D., Polewski, K., Rolewski, P., Plucin´ski, P., Syawin´ski, J., 2002. Spectroscopic studies on UVC-induced photodegradation of humic acids. Electron. J. Pol. Agric. Univ. 5, 1 – 12. Syawin´ska, D., Polewski, K., Rolewski, P., Syawin´ski, J., 2004. Effect of oxygen on UV-induced photodegradation of humic acid obtained from composted hulls of Juglans regia walnut. Int. Agrophys. 18, 159 – 166. Steelink, C., Tollin, G., 1966. Biological polymers related to catechol: electron paramagnetic resonance and infrared studies of melanin, tannin, lignin, humic acid and hydroxyquinones. Biochim. Biophys. Acta 112, 337 – 343. Struyk, Z., Sposito, G., 2001. Redox properties of standard humic acids. Geoderma 102, 329 – 346. Thompson, R.H., 1987. Naturally Occurring Quinones III. Recent Advances. Chapman & Hall, London. Tratnyek, P.G., Scherer, M.M., Deng, B., Hu, S., 2001. Effects of natural organic matter, anthropogenic surfactants, and model quinones on the reduction of contaminants by zero-valent iron. Water Res. 35, 4435 – 4443. Zepp, R.G., Schlotzhauer, P.F., Sink, R.M., 1985. Photosensitized transformations involving electronic energy transfer in natural waters: role of humic substances. Environ. Sci. Technol. 19, 74 – 81.