CHEMILUMINESCENCE IMAGING OF THE FORMATION AND PHOTODEGRADATION OF HUMIC ACID DERIVED FROM 5-HYDROXY-1,4-NAPHTHOQUINONE (JUGLONE)

Zbigniew Górski, Piotr Rolewski, Danuta Sławińskia, Janusz Sławiński

ABSTRACT

A new model humic acid (HA) have been synthetized from 5-hydroxy-1,4-naphthoquinone (juglone, J), an allelopathic compound occuring in leaves, nut shells, exsudates of roots and barks of Juglans genera. Aqueous solutions of J were autooxidised at different pH 6-10 with mineral matrices: bentonite, glass and silica. The very slow autooxidation reaction was monitored by means of chemiluminescence (CL) imaging and kinetics supported by a highly sensitive CCD camera Molecular Light Imager. In the course of autooxidation that lasted three weeks, an extremely low-intensity of CL was detected The reaction liquid was further treated according to the IHSS protocol to isolate humic acid fractions. Elemental analysis, UV-VIS and FTIR absorption spectra as well as fluorescence excitation and emission spectra were measured to characterise this new HA. Juglone-derived HA (JHA) in Na₂CO₃ was irradiated with UVC (254 nm) and induced changes in the UV-VIS absorption

Key words: juglone, toxicity, synthesis of humic acids, chemiluminescence, photodegradation.

INTRODUCTION

Litter of plants abundant in polyphenolic and quinone compounds can be a source of HS with specific, yet slightly recognized activities reflecting a high contents of quinone -hydroquinone redox systems [5,6,16,24]. The largest concentrations of quinones-hydroquinones, especially naphthoquinones such as juglone (5-hydroxy-1,4-naphthoquinone, and hydrojuglone (converted to juglone by sensitive plants) occur in the genera Juglans e.g. black walnut (J. nigra), European walnut (J. regia) and other closely related trees. Juglone and its derivatives are allelophatic phytotoxins - respiration inhibitors that deprive sensitive plants of needed energy for metabolic activity [2,5,25]. The soil and litter under the canopy of Juglans nigra or J. regia trees injures or kills many cultivable plants. Plants adversely affected by being grown near Juglans sp.trees exhibit symptoms such as foliar yellowing, wilting, and eventually death [27,28]. The questi on arises whether humified plants’ litters (composts) containing residues of Juglans sp. are also toxic ? The question apart from being of practical significance for horticulture in temperate and subtropical climates, is also vital in southern Poland where Juglans sp trees are commonly cultivated for valuable hardwood lumber and edible nuts. Moreover, humus-resembling polymers of quinones that strongly absorb electromagnetic radiation over a broad spectral range, particularly in the UV, contain a high amount of free stable radicals of a semiquinone type and exhibit both an oxygen- and light-dependent EPR signal and ultraweak chemiluminescence (CL) [14,19,20]. HA containing residues of naphthoquinones may be expected to interact strongly with the UV-VIS radiation and atmospheric oxygen that might modify toxic properties of composts and soils contaminated with juglone-rich plant materials. There has been no research on this subject so far.

As the first approach to solving these problems, we have studied the formation of HA-like macromolecular substances in the autooxidation process of juglone, and interactions of these substances (model J HA) with UVC radiation. Chemiluminescence (CL) imaging technique and spectroscopic methods were employed as the most adequate to the nature of the investigated processes.

MATERIALS AND METHODS

5-hydroxy-1,4-naphthoquinone (juglone, 97%) was purchased from Sigma. Porous silica SiO₂ while glass granules came from POCh, Gliwice, Poland. CL enhancer 3-amino-phthalhydrazide (luminol) was also from Sigma. Thanks to prof. dr A. Krysztafkiewicz, PUT, Poznan, we came into possession of bentonite. Such mineral matrices were used in order to simulate organic/mineral interactions alike the ones occurring under natural conditions. Other reagents were of analytical grade, water bidistilled. 0.1 mM juglone solutions in water, phosphate buffer pH = 8.3 and 10 mM Na₂ CO₃ pH = 10.4 alone and with SiO₂ admixture, bentonite or glass granules were exposed to slow autooxidation in air at 22 ± 1 °C in porcelain cuvettes inside a light-tight compartment of the chemiluminescent imager to monitor the intensity of CL.

To check whether CL from autooxidizing juglone and UV-irradiated juglone-derived HA is measurable we used a highly sensitive cooled (-70 °C) slow-scan charge coupled device (CCD) camera 'Night Owl' Molecular Light Imager LB 981 EG&G Berthold was applied. The spectral sensitivity of back illuminated photosensor lies in the region 180-1100 nm, resolution 512 x 512 pixels, quantum efficiency 40 % at 650 nm, as the first approach whereas for exposure times vary from ms to hours, readout and thermal noise achieve 6 e^- and 1 e^- /1000s, respectively. For standarization a radioluminescent spots of ⁶³Ni incorporated into porcelain was used. Data acquisition, and analysis and quantification procedures were carried out with the WinLight EG&G Berthold software (see details presented elsewhere [7,8]). Time-sequence series of CL-pseudocolored images from solutions of autooxidizing juglone at mineral matrices and solutions of synthetic HA from juglone expo sed to UV radiation were obtained.

Elemental analyses for C, H, N and S were measured with Vario EL II. Ash contents was determined by combustion (in case of HA derived from Juglans regia L. nut hulls compost). The results of the weight percentage determination are summarized in Table 1.

Absorption spectra were measured with a Top Sensor System from Ocean Optics (Netherlands) sensitive in the spectral range 220-800 nm. Quartz cuvettes 0.1 and 0.2 cm wide for UV and 1 cm wide for the visible range were used. Concentrations of juglone and HA ranged from 0.01 to 0.01mg/cm³ 10mM Na₂CO₃ adjusted properly to keep the absorbance range within ca 0.05 – 1.8. Absorbances A_{i, j} and colour coefficients Q_i/j = A_i / A_j, where i and j stand for selected wavelengths, typically 260, 400 and 600 nm , were calculated from the absorption spectra.

Infrared (FTIR) spectra of juglone and synthetized J-HA were measured on KBr pellets ( 3-6 mg / 5 mg KBr) by a Bruker IFS113V spectrofotometer. FTIR spectra of HA extracted from Juglan regia L. nut hulls compost are given elsewhere [22].

Fluorescence excitation and emission spectra were recorded with a Shimadzu RF 5001 PC spectrofluorimeter. Fluorescence of the control (‘dark’) and UV-irradiated solutions 10^-5-10^-4 g/cm³ of juglone and HA was measured at several excitation wavelengths covering the spectral range from 260 to 460 nm. Since 10mM Na₂CO₃ reveals a weak fluorescence in the spectral range 380-420 when excited at 260-300 nm, its fluorescence was subtracted from the total fluorescence signal.

UVC irradiation was performed using a low-pressure Hg Philips TuV115 VHO lamp at 10 cm distance from samples giving the total irradiation flux of 113 W/m² (27% at wavelength 254.7 nm). Irradiated solutions were immediately poured into dark-stored curettes and placed in the dark chamber CCD camera. The time interval between the end of irradiation and the start of imaging procedure was ca 3 min.

RESULTS AND DISCUSSION

Autooxitation of juglone to humic-like materials

At first, the dark count emission inside the chamber (the background emission or the aparature noise), ultraweak spontaneous emission from dry matrices, and the radioluminescent standard were checked. The background emission was found on average 77 counts per mm² per hour or 2.2 counts per second per cm². The maximum fluctuation of counts fell into the range 74-80 counts per mm² per hour and was caused mainly by technical imperfections of the thermal stabilisation system and ambient cosmic and telluric radioactivity. The values of the photon emission flux (surface emissiveness) from dry and dark-adopted matrices were almost the same as for background; the differences between them were not higher than ą5 photocounts/mm² h, i.e. they were statistically insignificant.

Next, dry matrices were conditioned 2 h with water, 0.1 M Na₂PO₄ and 10 mM Na₂CO₃. The emission intensity (count rate) from all matrices with solutions (see Photo 1, Fig.1) of 77-88 counts per mm² per hour, was found insignificantly higher than the dark current (background emission, 75-86). The highest value was recorded for SiO₂ + H₂O. However, SiO₂ is known to display a strongly delayed luminescence of a recombination character which can be affected by certain physicochemical factors, such as temperature or water [7]. Juglone solutions in water, phosphate buffer and sodium carbonate without mineral matrices were tested additionally. As it can be seen from Fig 1 and 2, the emission intensity from the all juglone solutions was slightly higher than from the background, but differences among the emission intensi ties from juglone in various solvents were small and at the threshold of statistical significance.

Autooxidation of juglone (J) solutions concomitant with the generation of P products (intermediates) in electronic excited states P* and subsequent photon (hn ni) emission (luminescence):

J + O₂ ¾®    P*   ¾® P + h ni

at three values of pH and three mineral matrices was performed in air inside a light-tight compartment of the 'Night Owl' CCD camera. A pseudo-colouring technique was applied to enhance the visual perception of small differences in the light intensity delta I emitted from the sample. Nevertheless, it proved inefficient to differentiate the images clearly. Therefore, kinetic curves of CL intensity I = f(t), (Fig.2) were constructed from the sequence of 28 separate images obtained during the experiment. 1 h time gate (exposure) was applied for each image. As seen from Fig. 2, the intensity of CL accompanying autooxidation was very low, ranging from 73-90 photocounts/mm² h that only slightly though regularly exceeded the background emission. The reproducibility of the emission intensity from the same kind of samples at different x-y positions accounted to less than ą 5 counts. Thus, no statistically significant differences were found bet ween the kinetic curves <I_CL>= f(t) despite different pH values of the solutions and different mineral matrices tested. A very low CL intensity of the autooxidized juglone observed in the experiments was attributed to at least two factors.

Firstly, 'soft' conditions of the oxidative polymerisation/condensation reactions of juglone to humus-like macromolecular substances. Juglone itself is chemically a relatively stable substance in slightly acidic or neutral environment. Chemical transformations leading to humus-like macromolecular substances occur only at pH >10 when the anionic form of juglone prevails. In a natural environment, a rapid browning and darkening hulls of J.regia L. and J. nigra L. nuts is observed, but it is probably the effect of tyrosinase- and peroxidase-enzymatic oxidation and addition reactions of naphthoquinones and polyphenols to plant proteins. Moreover, in soils and waters there are always traces of metal ions such as Fe (II, III), Mn (II, IV), Ti (II,III,IV), Zn (II) and clay-coloids that can catalyse oxidative polymerisation reactions. In our experiments neither juglone solutions in water nor in Na₂HPO₄ reacted with oxygen quickly enough to produce detect able Cl. It is known that the intensity of CL I is the product of the total quantum efficiency F and the rate W of the reaction limiting step (chemiexcitation) in the generation of electronic excited states:

I = F W

The total quantum efficiency depends on the luminescent properties of the excited and ground-state products, and whereas luminescence-quenching properties of/on the medium. Low values of the luminescence quantum yield of quinones and their susceptibility to the singlet-triplet convertion do not facilitate high values of F. Thus, both factors F and W can contribute to low values of I. Even the rate of autooxidation in Na₂CO₃ at pH=10.4 is very low and an efficient yield of juglone-HA-like product requires the reaction time of several weeks. Surprisingly, mineral matrices do not accelerate these reactions.

Secondly, certain polymerization reactions proceeding by the radical mechanism have low free enthalpy delta G. Consequently, the probability of generating excited states energetic enough to produce photons with energy E= h ni > delta G (free or Gibbs’ energy of a reaction) in the spectral range 200-800 nm is extremely low. Moreover, HAs formed from quinones are known to quench luminescence of many organic compounds [15]. On the contrary, a relatively strong CL was observed during the oxidation of 1,2,3-trihydroxybenzene (pyrogallol) at aerated suspension of Al₂O₃ [10]. However, the reaction with pyrogallol is much faster than juglone autooxidation and proceeds through specific intermediates like purpurogallin [18].

Chemiluminescence in the photooxidation of juglone-HA

In order to check whether the UVC radiation can stimulate ultraweak CL of juglone-derived HA concomitant with the oxidative degradation, the experimental arrangement with eight curettes was set up (Photo 2a). Single photocount pseudocoloured images are shown in (Photo 2b). It can be seen that even 5 min long irradiation time stimulates a weak CL flux of about 80 photocounts/mm² h. It means that the absorbed UVC energy initiates slow exergonic reactions that generate electronically excited molecules P*, part of which undergoes a radiative deactivation (CL) and emits photons h ni’:

JHA + h ni UVC    ¾®   P* + h ni’

a)

b)

It is clear then that longer UVC irradiation times of 10, 30 and 60 min decrease CL intensity. This result indicates a high photoreactivity of the model jHA and its low photostability. Therefore, such a substance is likely to sensitise degradation processes instead of protecting against UV radiation. This inference is clearly relevant to the practicable photodetoxication of J regia – derived composts. The kinetic curves I = f(t) (Fig.3) are shown for comparison in two scales: counts/mm² h and number of photons/ mm² h since the spectrum of ultraweak CL is unknown and cannot be determined at this level of CL intensity. In further experiments when a strong CL enhancer is added, such a comparative representation may be useful both for recalculations and better understanding of the observed processes.

It is very likely that quinoid moieties play a crucial role in both primary photoreactions and chemical excitation leading to CL since:

1. Quinones absorb in the UV and blue part of the visible spectrum (see the absorption spectrum in the next chapter). It was shown that HA excited at 480 nm produces singlet oxygen ¹O₂* [12,20,21].

2. An efficient intersystem crossing singlet-to-triplet S* ? T* occurs in quinones (Q) [17,19]. Due to a longer lifetime in the T*-state, a higher probability of the reaction of ³quinone* with ³O₂ occurs which produces excited molecular oxygen molecules:

³Q + ³O₂ ¾® ¹Q + ¹O₂* (¹ delta_g, ¹ sigma_g⁺)

3. Light induces a transient increase in the EPR signal of Juglans regia-derived HA, i.e. increases a pool of unpaired electrons [14]. Paramagnetic moieties with unpaired spins in HA are susceptible to the reaction with triplet oxygen molecule. The kinetics curves I = f(t) of CL reflect slow oxidative degradation reactions initiated during the irradiation except the short initial time period of ca 10 min subsequent to UVC-irradiation. In this initial phase the overlapping of the delayed photoluminescence and CL can occur. I = f(t) curves for t > 10 min reflect a pure CL processes. The dynamic range of the CL decay and fluctuations in I-values do not allow to apply any reliable fitting procedure to neither the exponential or other character of CL kinetics.

In order to enhance the CL signal and gain information on the mechanism of HA-UVC interactions luminol, a well known enhancer of CL, was used in several combinations. Photo 3 and Fig. 4 illustrate the CL intensity and its kinetics for cases when luminol was added instantly after the irradiation. All the other parameters were the same as in the previous experiment. In all experiments with luminol the intensity of CL is calculated in abosolute units: photons per square mm per hour. It seems an appropriate unit, since the emitted light comes predominantly from the luminol- reaction product; aminophthalic acid which exhibits maximum of its fluorescence at 425 nm. A strong and well differentiated CL ranging from 850 to 5000 photons/mm² h depending on for different / which depends on irradiation times, i.e. on the absorbed dose of UVC radiation is evident. Comparison of CL from the control solution of 10mM Na₂CO₃ alone (cuvets g-j in Photo 4) and HA + Na₂CO₃ (a-f), clearly indicates that UVC radiation induces a distinct emission from Na₂CO₃. Indeed, a pseudocolouring representation of the superficial CL intensity corresponds to a higher photon flux than the one of the orange-red colour of the cuvette c. A closer examination of the UVC-induced CL from the irradiated Na₂CO₃ solution confirms this conclusion (Fig. 4). The initial (maximum) intensity exceeds 1800 photons/mm² s, thus it is higher by 20% than the one of the HA + Na₂CO₃ system. The first question arises then: what causes CL of Na₂CO₃ solution. On the base of earlier research [1, 26] and data shown in Fig. 4 and 5 it seems reliable to claim that Na₂CO₃ solution irradiated with UVC reveals much weaker CL than the one s hown in Photo 2 (without luminol). Moreover, luminol added after irradiation evokes strong emission (CL). Such results can be attributed to the energy of the UVC with 254.7 nm wavelength equal to 5 eV or 480 kJ/mol that can cause either a scission of C-O and O-H bonds (378 and 461 kJ, respectively) or ionization of carbonate anions. These processes lead to the formation of O₂^- and peroxalate or percarbonate, both known to be weak chemiluminogenic compounds. However, they easily react with luminolor, another strong fluorescing compound, that results in a strong CL. Prolonged irradiation (2 hours) decomposes peroxy-compounds and therefore the enhancement of CL from a long-time irradiated Na₂CO₃ solution (Photo 3 and 4, Fig. 4 and 5) is not observed. This interpretation accounts for the experimental data.

a)

b)

In another experiment performed with luminol added 1 h after the end of irradiation (Photo 3 and 4, Figs 4 and 5), a slightly lower CL intensity (photocount flux) from Na₂CO₃ + HA solution than from Na₂CO₃ solution was observed. It indicates that HA admixture reduces the CL intensity by:

1. a self-absorption effect,

2. decreasing the rate of physicochemical interactions leading to the formation of electronically excited molecules, and

3. a combined effect of the both processes.

Indeed, HA have been proved recently to be efficient free radicals scavengers and quenchers of CL [4].

Absorption spectra

Absorption spectra of juglone in aqueous solutions at various pH and juglone-derived HA are presented in Fig. 6. At pH > 10 a strong absorption band with maximum around 524 nm appears. It can be reversibly turned back to yellow colour by acidification. It means that the juglone molecule is ionized at pH > 10. A deep pink alkaline air-saturated solution of juglone at room temperature slowly darkens. After 3-4 weeks by decreasing pH to ca 2 a 'model' water-insoluble parahumic substance can be precipitated. The yield of the precipitate is ca 87%. As plotted in Fig.6, a weak shoulder in the absorption spectrum at ca 520 nm resembling the spectrum of the ionic form of juglone, is still observed. It might be interpreted as a 'remnant' of a juglone skeleton conserved in a macromolecular ensembles of HA.

It is worth mentioning that the 524 nm band is also slightly marked in HA prepared from the compost of green nut hulls Juglans regia L. A slow rate of juglone oxidative polymerization/condensation and transformation into parahumic substances observed by absorption measurements seems coherent with a very low CL intensity recorded in previous chemiluminescence imaging experiments (Photo 1 and Figs 1 and 2).

Strongly regular trends in FTIR spectra of juglone (j), model juglone-derived HA (j-HA) and HA isolated from compost of J.regia (com-J) were observed. The most dramatic changes are recorded in the ratio of relative intensity of absorbancy in the wavenumber region 500-1750 cm^-1 to the one of 2250-3700 cm^-1 , namely j : j-HA : com-j = 7.0 : 3.4 : 1.0. In this order a new absorption peak with the wavenumber at ca 3380 cm^-1 grows and increasing overlapping of different oscillations in the region 500-1750 cm^-1 is observed. Such an evolution of FTIR spectra indicates a gradual increase in the complexity of HA macromolecules and their intramolecular couplings. The detailed analysis of FTIR spectra will be will be dealt with in a separate paper.

UVC irradiation causes absorbancy to decrease with the irradiation time and brings changes in the color coefficients Q_i/j within the whole wavelength range (Table 2). Solutions irradiated longer than 40 min and subsequently treated with HCl (pH=1-2) do not precipitate HA-like pellets. The results prove that HA undergoes UVC-stimulated oxidative degradation (photobleaching) to water-soluble fractions – fulvic acids, with likely progressing reduction of their molecular size and further degradation to more hydrophillic compounds. Colour coefficients Q_i/j of HAs are known to be correlated with molecular weight (the correlation coefficient r »0.80) as well as with the contents of both phenolic hydroxyl group and atomic N/C ratio (r » 0.6-0.5, respectively) [3, 23]. Results of previous research [13] pointed to o- and p-quinone moieties in HA as the weakest arrangements with strongly pol arized carbonyl groups >C = O and a low electron density between the adjacent C-C bonds in the ring. It is correlated with the increase of the Q _2.6/4 and Q_4/6 –values (Table 2). Quinones have a long wavelength absorption band in the range 380-440 nm and their degradation produces carboxylic acids and phenolic compounds absorbing at ca 250-280 nm.

Fluorescence excitation and emission spectra

Fluorescence emission spectra of juglone solutions recorded during the autooxidative polymerisation (model 'humification') to HA-like macromolecular substances at various pH, concentrations and the excitation wavelengths 260, 280, 300,320, 340, 360, 380, 400, 420 and 440 nm were recorded. Additionally, in order to introduce necessary corrections, fluorescence from buffer solutions was recorded. The corrected and concentration-optimised fluorescence emission spectra are shown in Fig 7. The excitation at 260 nm gives too large contribution to fluorescence emission from sodium phosphate and sodium carbonate, even after recrystallisation. The emission spectra taken at 350, 440 and 460 nm excitation wavelengths give reliable results. The maxima observed at the shortest emission 435-460 nm (excitation at 350 nm) may be attributed to fluorescence from phenolic groups conjugated to carbonyls through the benzene ring. The emission peaks in the region 440-470 nm may be ascribe d to hydroxycoumarin-like structures and substituted phenolic and naphtholic subunits. Formation of coumarin-like structures (1,2-benzopyrone) from anions of 5-hydroxy-1,4-naphthoquinone and neutral juglone seems likely. Coumarins are efficient fluorescers in the spectral region 440-500 nm.

A general feature observed is that the wavelength of the maximum emission increases with the excitation wavelength. This tendency has been also observed for many different HA by other authors. The spectra recorded here are typical for HA and may be attributed to highly substituted aromatic nuclei with at least one electron-donating group and/or to conjugated unsaturated moieties of a high degree of resonance [3,6,11]. It confirms the conclusion that the material obtained from juglone resembles natural 'jung' humic substances. The second finding, an enhancing effect of ionization upon the intensity of fluorescence, has been also observed for other HA [3,9,15]. In our case concentration - intensity dependence was of a non-linear character. All the data demonstrate intrinsic properties of humic substances and indicate that energy transfer processes, inner filter effects and quenching may have a significant influence upon fluorescence properties of juglone-derived HA. Such properties are ty pical for HA and therefore the product obtained from juglone may be considered as a model of low-polymerized parahumus substances.

The effect of UVC irradiation time (t_ir) on the fluorescence spectra is also seen in Fig.8. The shape of spectra and position of the emission maximum does not change significantly during irradiation, though values of the amplitude increase with t_ir progressing. It can be seen that the short wave excitation 260 and 350 nm leads to a gradual increase in I_max, while the long-wave excitation 440 nm reveals saturation, and lambda excitation 460 nm gives a clear maximum at I_max= f(t_ir) after t_ir of ca 60 min. This result indicates that long-wave fluorophores are degraded faster than the short-wave absorbing/emitting ones and degradation of the former produces short-wave fluorophores. The structure of such fluorophores should be simpler than in initial absorbers since the absorbance of the irradiated solution decreases with t_ir. It is very likely that the light-excited naphthoquinone arrangement s undergoe ring-opening reactions resulting in formation of species that absorb at shorter wavelengths. Correlation with the Q_2.6/4 colour coefficients increase during UVC irradiation. Attributing the effect to faster degradation of quenching subunits in the vicinity of the short-wavelength or to photodegradation of HA that leads to the coil-chain transitions of the macromolecular complex with less compact structure and better exposure of fluorophores to excitation seems less plausible interpretation.

All these results lead to the conclusion that HA isolated from composted green hulls of Juglans regia L. nuts is sensitive to UVC radiation and undergoes photochemical oxidative degradation to small-molecular and more hydrophyllic compounds. Possible agricultural and environmental aspects of these findings will be shortly discussed further.

CONCLUSIONS

Chemiluminescence imaging, absorption and fluorescence spectroscopy was applied to study autooxidation of 5-hydroxy-1,4-naphthoquinone (juglone) to model humic-like substances for the first time. The CL imaging technique was applied to detect and quantify the generation of electronically excited states during the autooxidation/polymerisation (model humification) of juglone at pH = 6-10 at mineral matrices. The sequence of pseudocoloured SPC images and kinetic curves <I_cl> = f(t) revealed very slow reactions rate with reactions lasting several weeks and resulting in the formation of HA-like products. An extremely low quantum yield and intensity of CL in these complex reactions was observed. Surprisingly, mineral matrices: glass, porous silica and bentonite did not show statistically significant effect on the intensity of CL and the 'humification' rate. In contrast to it, UVC-induced photodegradation of synthetic HA produced CL indicating the generation of electronica lly excited states with the energy ca 1- 5 eV. A degradative character of the reactions was confirmed both by the observed transient increase in fluorescence intensity that decreased subsequently, and the decrease in absorbancy of HA within the whole measured spectral range (240-700 nm), as well as the changes in colour coefficients Q_2.6/4 and Q _4/6. The application of a chemiluminescent probe-enhancer such as luminol, which reacts with peroxyradicals, not only did amplify the ultraweak CL signals but also proved the involvement of ROS in photoreactions and consecutive post-irradiation (dark) degradative processes. Quinone moieties of HA are the most probable target of such degradative photoreactions. Fulvic-like acids and even simpler low-molecular hydrophyllic compounds, mostly aliphatic, are expected to be final reaction products.

At least two consequences, vital in agricultural and ecological terms are pertinent to the obtained results. Namely, heavy in consequences a global-scale impact of photooxidative degradation of HA and, more particular, potential allelopathic effects of biomaterials containing juglone and its derivatives.

The first one increases a green-house effect, acidification of soils and surface waters, brings changes in sorption capacity and hormon-like physiological activity of humic substances and increased availability of organic carbon for soil/water microorganisms. Due to the second one UV-induced photoreactions involving susceptible quinone arrangements might be beneficial to detoxification of litter and composts containing allelopathic phytotoxins derived from hydroxynaphthoquinones occuring in Juglans gender.

The research was supported by grant 3OP4G 016 22 from the State Committee for Scientific Research, Poland.

REFERENCES

1. Albrecht S., Hornak H, Freidt T, Boehm W.D, Weiss K, Reinschke A. 1993. Chemiluminometric measurement of oxalate. J. Biolumin. Chemilumin., 8, 221-24.

2. Babich H., Stern A., 1993. In vitro cytotoxicities of 1,4-naphthoquinone and hydroxylated 1,4-naphthoquinones to replicating cells. J Appl. Toxicol., 13 (5) 353-358.

3. Chen J., Gu B., LeBoeuf E.J., Pan H., Dai S., 2002. Spectroscopic characterization of the structural and functional properties of natural organic matter fractions. Chemosphere, 48, 59-68.

4. Chmura J., Sławinski J. 1998. Humic acids and melanins as scavengers of electrochemically generated peroxyradicals. Curr. Topics Biophys.. 22(B) 46-51.

5. Dana M.N., Lerner B.R. 1994. Black walnut toxicity, Gen. Horticulture HO-193, 1-2,

6. Drozd J., Gonet S.S., Senesi M., Weber J., 1997.The role of humic substances in the ecosystems and environmental protection. PTSH-Polish Soc. Humic Substances, Wroclaw, 596-602.

7. Gorski Z,. Slawinski J.: 2001. Chemiluminescence imaging of humus acids interactions with UV radiation and ozone. In: Chemiluminescence at the turn of the Millenium. S. Albrecht, T. Zimmermann, H. Brandl, eds. Schweda Werberdruck GmbH, Dresden, 53-58.

8. Gorski Z., Slawinski J.: 2002. Use of a CCD-monitored chemiluminescence imaging for sensing of the UV- and ozone-induced degradation of humus substances. Proc. SPIE: 4887, 23-31.

9. Hautala K, Peuravuori J, Pihlaja K., 2000. Measurements of aquatic humus content by spectroscopic analyses. Water Res. 34(1): 246-258.

10. Kroh J. 1963. Chemiluminescencja w fazie ciekłej [Liquid Phase Chemiluminescence]. ŁTN, wydz III, Łódź [in Polish].

11. Leenheer J.A., Wilson M.A., Malcom R.L., 1987. Presence of potential significance of aromatic-ketone groups in aquatic humic substances. Org. Geochem. 11,273-277.

12. Paul A, Hackbarth, Roeder B and Steinberg CEW. 2003. Photogeneration and quenching of singlet oxygen by humic substances: A spectroscopic study. 10^th Congress Eur.Soc.Photobiol, September 6-11,2003, Vienna, PO22, p.85.

13. Polewski K., Sławinska D., Sławinski J. 2002. The role of ortho- and para-semiquinones in the chemiluminescence and antioxidizing activity of humus substances. In: Stanley P., Kricka L.J. Bioluminescence and chemiluminescence Progress and current status. World Scientific, New Jersey, Singapore, 161-164.

14. Polewski K., Sławinska D. and Sławinski J. 2003. EPR study of photoinduced changes in natural humic substances. 10^th Congress Eur.Soc.Photobiol, September 6-11, 2003, Vienna, PO12, p.84.

15. Puchalski M,M, Morram M, J. and Von Wandruszka R. 1992. Fluorescence quenching of .synthetic organic compounds by humic materials. Environ. Sci. Technol. 26:1787-1792.

16. Rietveld W.J. 1983. Allelophatic effects of juglone on germination and growth of herbaceous and woody species. J. Chem. Ecol. 9: 295-308.

17. Scott D.T., Mc Knight D.M., Blunt-Harris E.L., Kolesar S.E., Laley D.R., 1998. Quinone moieties act as electron acceptors in the reduction of humic substances by humic-reducing microorganisms. Environ. Sci. Technol. 32, 2984-2986.

18. Sławinska D., Lichszteld K., Michalska T., 1978. Singlet oxygen and chemiluminescence in autooxidation of pyrogallol. Pol. J. Chem. 52, 1725-1737.

19. Sławinska D., Sławinski J., Sarna T. 1975. Photoinduced chemiluminescence and EPR signals of polyphenol- and quinone-polymers. Photochem. Photobiol., 21: 393-397.

20. Sławinska D., 1978. Chemiluminescence in the formation of singlet oxygen in the oxidation of certain polyphenols and quinones. Photochem. Photobiol. 28, 459-463.

21. Sławinski J., Puzyna W., Sławinska D., 1978. Chemiluminescence during photooxidation of melanins and soil humic acids arising from the singlet oxygen mechanism. Photochem. Photobiol. 28, 459-463.

22. Sławinska D , Polewski K, Rolewski P, Plucinski P. and Sławinski J. 2002. Spectroscopic studies on UVC-induced photodegradation of humic acids. Electr. J.Polish Agric. Universities, 5(2) 1-10.

23. Tan K.H. Humic matter in soil and the environment, principles and controversies. Marcel Dekker Inc., N.Y. 2003.

24. Thompson R.H., 1987. Naturally occurring quinones. III. Recent Advances. Chapman and Hall, London.

25. Whittaker R. H., Feeny P.P., 1971. Allelochemicals: Chemical interactions between species. Science, 171, 757-770.

26. Wierzchowski..J., Sławinska D., Sławinski J. 1986. Carbonate and superoxide ion-radicals as intermediates in the chemiluminescence of the ferricyanide-hydrogen peroxide redox system. Z. Phys. Chem, Neue Folge, Munchen, 148: 197-214.

27. www.ansci.cornell.edu/courses/as625/1999term/boyer/juglone.html

28. www.ansci.cornell.edu/courses/as625/1999term/boyer/

Zbigniew Górski, Janusz Sławiński
Department of Radio- and Photochemistry
Institute of Chemistry and Technical Electrochemistry
Poznan University of Technology
Poznan, ul. Piotrowo 3, 60-965 Poznan, Poland
e-mail: zbigniew.gorski@put.poznan.pl

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed ‘Discussions’ in each series and hyperlinked to the article.