A study of the photosensitizing properties of seawater

Marine Chemistry, 12 (1983) 1--14 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

1

A STUDY OF THE PHOTOSENSITIZING PROPERTIES OF SEAWATER A. MOMZIKOFF

Institut Oc~anographique, Laboratoire de Physiologie des Etres Matins, 195 Rue SaintJacques, 75005 Paris (France) R. SANTUS

Laboratoire de Physico-Chimie de l'Adaptation Biologique, Museum National d'Histoire Naturelle, 43 Rue Cuvier, 75231 Paris C~dex 05 (France) M. GIRAUD

Laboratoire de Chimie Organique, Museum National d 'Histoire NatureUe, 63 Rue Buffon, 75005 Paris (France) (Received December 8, 1981;revision accepted June 21, 1982)

ABSTRACT

Momzikoff, A., Santus, R. and Giraud, M., 1983. A study of the photosensitizing properties of seawater. Mar. Chem., 12: 1--14. The naturally occurring photosensitizers present in the Dissolved Organic Matter (DOM) of seawater are able to promote photodynamic reactions and, specifically, singlet oxygen formation. Using riboflavin, a constituent of the DOM, as a reference photosensitizer, it is shown that in the concentration conditions normally encountered in seawater, the concentration of singlet oxygen is proportional to the photosensitizer concentration which makes it possible to define a photosensitizing efficiency (PSE) of seawater samples. The distribution of the PSE values obtained from samples collected at different stations (off-shore, coast) and at different seasons, has been measured. Comparison of the profiles indicates that the PSE decreases with depth for filtered and unfiltered water and that the distribution of the PSE is notably influenced by weather conditions in the upper 10m and especially at the air--sea interface.

INTRODUCTION

Light penetration into the euphotic layers of the ocean is one of the essential factors controlling the development of the algal biomass and consequently processes which depend on it such as the fluxes of organic compounds between living organisms and dissolved organic matter (DOM). Many studies have been devoted to the study of photosynthetic reactions in marine phytoplankton and also to the research on compounds assimilated or excreted by phyto- and zooplankton. Over the last ten years, our knowledge of the chemical composition of DOM has regularly increased. Despite the fact that DOM concentration is roughly ten times higher than that of the amount of living matter found in the sea (expressed in carbon units), only few data are at present available on the in situ chemical evolution of the dissolved compounds under physical factors and especially 0304-4203/83/0000--0000/$03.00

© 1983 Elsevier Scientific Publishing Company

under the influence of light. As examples, in two recent reviews, Zafiriou (1977) and Zika (1981) emphasized the necessary researches to be undertaken in this field. Studies on the photochemical evolution of the DOM of natural waters develop more rapidly for fresh-water than for seawater, where analytical problems arise because of the high salt concentration. Zepp et al. (1977), Wolff et al. (1981) and Zepp et al. (1981) showed that singlet oxygen (l O~ ) was formed in river and estuarine waters upon photosensitization with humic acids of terrestrial origin. In addition, Joussot-Dubien and Kadiri {1970) pointed out the possible formation of singlet oxygen in seawater by photosensitizers such as riboflavin. As shown by one of us (Momzikoff, 1977), flavins and pterins which absorb in the near u.v. (Albert and wood, 1953; Landymore and Antia, 1978) may be considered, among other dissolved fluorescent substances, as a constituting fraction of the DOM. Moreover, since pterins (Chahidi et al. 1981; Momzikoff and Santus, 1981) and ftavins are well recognized photosensitizers, it was of interest to measure their photosensitizing properties in conditions not too far removed from those encountered in seawater and compatible with classical colorimetric or fluorimetric methods for evaluating their possible in situ photochemical roles, especially as singlet oxygen producers. In this article we first compare the photosensitizing properties of riboflavin (RF) and a series of eight pteridine derivatives found in seawater or known to be excreted by living organisms. Then, in a second part, we compare the photosensitizing efficiencies (PSE) of samples of Mediterranean seawater. In both cases, an aromatic amino a c i d - Tryptophan ( T r p ) - commonly used for evidencing photodynamic reactions in aqueous solutions (Walrant and Santus, 1974), has been chosen as a reference substrate for illustrating these photosensitizing potentialities.

MATERIAL AND METHODS

Photosensitizers

Beside riboflavin, a Merck product, eight pteridine derivatives were studied: (1) pterin (R1 = R2 = H), (Fluka); (2) xanthopterin (Rl = OH, R2 = H), (Fluka); (3) isoxanthropterin (Rl = H, R: = OH), (Fluka); (4) 6-carboxypterin (Aldrich); (5) 7-carboxypterin (Aldrich); (6) biopterin: L-erythro-6 (1', 2', 3'-trihydroxypropyl) pterin (supplied by Professor M. Viscontini); (7) 7-hydroxybiopterin (supplied by Professor R. Tschesche); (8) 6-carboxy-isoxanthopterin (supplied by Professor E.C. Taylor), The structure of these chemicals is shown in Fig. 1.

O H N ~ N ' ~ O~,,.N~

CH3

Nf ~,,,~ --CH3

I

Ribose (a)

O

O

NN N R

H (b)

(c)

Fig. 1. Chemical structure of: (a) riboflavin, (b) lumazin, (c) pterin (RI and R2 are given in the text).

0 ther reagents FeCI3 (Merck); diatom earth (Prolabo) and Kaolin (Prolabo) were thoroughly washed before use, first with 5 N HC1 then with large volumes of water to bring the suspension back to neutrality.

Tryptophan determination A 2 x 10 -s M stock solution of Trp (Calbiochem) in seawater was prepared. The Trp concentration was directly assayed in seawater according to the Escande et al. method {1977}. At the concentration used, in the presence of formaldehyde and hydrogen peroxide, Trp was quantitatively converted into norharmane which was fluorimetrically assayed (excitation, 386nm; fluorescence, 477nm) after blank correction.

Seawater samples Artifical seawater was prepared according to Lyman and Fleming (1940). Natural samples were collected in the Mediterranean Sea. Station A was located 6 nautical miles from Monaco (43 ° 41' I"N; 7 ° 26' 5"E) on a 500m bottom. Station C was located in a small hay near Calvi (Revellata), Corsica. Collection of samples was performed in May 1980 (Stations A1 and C) and in September 1981 (Station A2 ). Station R was in Baie de Roquebrune, 0.5 nautical miles from the coast (bottom varying from 25 to 30 m); samples were collected in September 1981 in a choppy sea (Force 3) over several days. A 1-1 polyearbonate reversing bottle (Mecaboller) was used for collecting deep-sea samples and a Garrett sieve for the air-sea interface film (Garrett, 1965).

The uncorrected sample temperatures are given in Figs. 5--8. Water samples were filtered through glass-fibre filters (Whatman GF/C) and frozen until studied. Irradiations

Two types of quartz cuvettes were used: a 20-ml one ( l c m wide, 5 c m optical pathlength) or a 5-ml one (1 × 1 x 5cm). They were placed 8 c m in front of a 1000W Oriel Xe--Hg lamp. Samples were thermostatted at 15°C + 0.5. Wavelengths shorter than 3 2 0 n m were removed by using a Pyrex glass optical filter. Solutions were bubbled with either air or nitrogen supplied b y Air Liquide. Irradiations aimed at comparing the PSE of the synthetic sensitizers in artificial seawater were performed in the 20-ml cuvette. The solution to be irradiated was obtained by mixing an equal volume of the photosensitizer solution (2 x 10 -6 M) and the Trp solution (2 × 10 -5 M). The initial Trp concentration was thus ten times higher than that of the sensitizer, the initial volume being 18ml. Aliquots of l m l were taken after 1, 2, 3, 5, 10, 20 and 30 min of irradiation. Undestroyed Trp was assayed as described above. The 5-ml cuvette was used for measuring the variations of the amount of Trp destroyed as a function of the R F concentration. Equal volumes of Trp (2 x 10 -s M) and R F solution were mixed in order to adjust the final Trp concentration to 1 0 - S M and the R F concentrations to 10 -9, 10 - s , 3 x 10 - s , 6 x 10 - s , 10 -7 , 3 x 10 -7 or 1 0 - 6 M , respectively. For each R F concentration the kinetics of the Trp destruction was followed b y taking aliquots after 1, 2, 3, 4, 5, 6, 7 and 8 min of irradiation. When dealing with seawater samples, 0.5ml of a 2 × 1 0 - 4 M Trp solution was added to 10ml of the melted sample (final Trp concentration 10 -s M) and 3.2ml were irradiated for 5 min in the 5ml cuvette. RESULTS AND DISCUSSION

Because of this new approach to DOM chemistry, it seems appropriate to us to give some generalities on photochemical reactions. Such a reaction begins when a molecule in its ground state absorbs one p h o t o n in the u.v./ vis. range of the solar spectrum corresponding to its absorption band. The electronically excited molecule is brought to a short-lived (10 -9 s) singlet excited state which can deactivate either radiatively (fluorescence) or nonradiatively to the ground state or undergo intersystem crossing conversion to the longer lived (ps to ms} triplet state. This excited molecule can undergo photolysis or intramolecular rearrangements, such reactions being called 'direct photochemical reactions'. On the other hand, when the molecule in its triplet state transfers its energy to another molecule, the reaction is said to be 'photosensitized' and the chromophore is called a photosensitizer. When

the photosensitized reaction requires oxygen, one deals with a photodynamic reaction. There are two types of photodynamic reactions. In the case where the acceptor of the triplet energy is molecular oxygen (O2), singlet oxygen (102), a highly reactive species, is formed; the photosensitized reaction is said to be Of Type II (Gollnick, 1975). A Type I reaction occurs when the substrate is directly oxidized by the photosensitizer triplet state -- other activated oxygen species (superoxide ion O~-) may be produced during the course of Type I processes. These Type I reactions require a high substrate concentration for high efficiency. It thus clearly appear that, in view of the rather high oxygen concentration (at least 1 x 10 -4 M) and the low DOM concentration (a few mg per liter), only Type II photodynamic reactions must be considered in the upper layers of the ocean.

Influence of the photosensitizer Figure 2 shows the typical kinetics of the Trp destruction photosensitized by RF and the eight model pteridines in aerobic conditions under white light irradiations: the first part of each curve may be approximated by a straight line and represents the initial velocity of the Trp destruction induced by the various sensitizers under study. No significant destruction occurred in nitrogen-saturated solutions, confirming the photodynamic nature of the photosensitized Trp destruction. The same experiments performed under nearly monochromatic irradiation (i.e. 325 < k < 335nm) leads to similar kinetics and shows that the rate of 100

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Fig. 2. Variation of the Tryptophan (Trp) concentration (expressed in percent) as a function of the time of irradiation ()~ ~ 320nm) in presence of various photosensitizers in artificial seawater. Concentration conditions are given in the text. (1) Riboflavin, (2) biopterin, (3) 7-carboxypterin, (4) pterin, (5) lumazin, (6) xanthopterin, (7) isoxanthopterin, (8) 7-OH-biopterin.

Trp destruction is practically the same using either biopterin or riboflavin as sensitizers, provided one deals with solution of equal absorbance at the irradiation wavelength. In such conditions, the quantum yield for the photo~ dynamic reaction is found to be 0.01 (Momzikoff and Santus, 1981) using the ferrioxalate actinometer as quantum counter (Parker, 1968) and conse. quently the apparent J O2 formation quantum yield as measured, using Trp as a 102 probe is (1,l O 2 ~ 0.01. This value is consistent with the one reported by Chahidi et al. (1981) in which the higher 102 yield (0.04) was measured using high histidine concentrations, oxygen saturation (10 -a M) and pterin as sensitizer. It is well known (Gollnick, 1975; Pileni et al., 1978) that k[S]

(1)

where k is the bimolecular rate constant for I O 2 scavenging, T the 1 0 2 lifetime (~ 3ps in water) and S the substrate. The use of Trp as a 102 probe requires justification since Trp is known to be a substrate for both Type I and Type II photodynamic reactions (Boutdon, 1972). However, as described above, Type I reactions can be neglected for concentration reasons in euphoric sea layers. Moreover, this amino acid does not absorb in the wavelength range normally encountered in photosensitizers (/> 320nm) and thus, in contrast with other 1O2 probes such as 1, 3
Influence of mineral particles and metal ions In order to simulate conditions which may be encountered in some marine envirionments, we have examined the effects of the addition of

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Fig. 3. Variation of the Trp concentration (expressed in percent) under irradiation with k/> 320nm of artificial (1, 2, 3) and natural (4, 5, 6) seawater samples. Concentration conditions are given in the text. mineral particles and metal ions on the PSE of model photosensitizers. From our results (Fig. 3) it m a y be concluded that diatom earth, kaolin or Fe 3 + (data not shown) at concentrations (10 -6 M) largely above those found in sea water do not appreciably affect the Trp photo-oxidation kinetics. These results may be partly explained by the low affinity of the pteridine toward these absorbents and the lowering of t h e metal ion-pteridine affinity constant because of the ionic strength of seawater. In the absence of a complex formation, the low metal ion concentration used in the present experiments impedes any deactivation of the excited singlet or triplet state of the photosensitizer by the paramagnetic metal ion.

Influence of the photosensitizer concentration An important parameter which characterizes seawater is the photosensitizing efficiency (PSE) which can be defined as the potency of a sample to promote, under standard irradiation conditions (e.g. a given irradiation time under identical excitation wavelengths), the destruction of a substrate via photodynamic reactions. It is of course a relative valise which makes it possible to compare various samples without introducing the quantum yield, a concept of little value in seawater samples. Thus, the uncertainty and t h e broad variations in the nature and the concentration of the photosensitizers present in seawater explain the impossibility of determining absolute quantum yields. Among the photosensitizers to be found in seawater, one can cite flavins, pterins, degraded chlorophylls (pheophytins),

humic acids and aromatic hydrocarbons (resulting from pollution), all of them good 102 producers (Foote, 1976). In addition, it can be expected that the most hydrophobic chromophores would be concentrated at the air-water interface. The calculation of the PSE of seawater samples requires the determination of irradiation conditions for which the ~O2 steady state concentration (and thus the PSE) is proportional to the absorbed light. As evidenced in Figs. 2 and 3, at long irradiation times (large Trp destruction), the photosensitized reaction reaches a plateau where any PSE determination would be meaningless. Hence, it is well known that at low chromophore concentration (weak optical densities) the exponential Beer--Lambert light absorption law can be approximated by a linear function. In these conditions, the 1O: steady-state concentration is expressed as (see for instance, Walrant (1976) and references therein) [1 0 2

] CCCT

Io(~)ecl[02 ]

(2)

Where I0~) is the incident intensity of the polychromatic light (in einsteins cm-2 s-1 ), CT the triplet formation quantum yield of the photosensitizer and [O: ] the oxygen concentration in seawater. Equation 2 indicates that the percent of Trp destruction which is proportional to the light absorbed during the irradiation duration (At): I0(x) e(x)clAt, is therefore proportional to the concentration (c) of the photosensitizer having a molar extinction coefficient ec~) at wavelength X. .......................................

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Linear relationship between the percentage of Trp destruction and the r i b o f l a v i n concentration in artificial seawater after various times o f irradiation. Fig. 4.

9

This is illustrated in Fig. 4 which shows, for irradiation times up to 5 minutes, the good proportionality of the Trp destruction with the concentration of RF used as model photosensitizer under irradiation with wavelength >/320nm.

Application to seawater samples In seawater samples where n photosensitizers with triplet formation quantum yield ~Wi and molar extinction coefficient ei(X) at wavelength X are present at concentration c/, one has i=n

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Fig. 5. Station A 1 ( M a y 1980). Variation of the temperature (dashed line) and the PSE (solid lines) as a function of depth for filtered (b) and unfiltered (a) seawater.

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Fig. 8. Same as Fig. 5 except that samples were collected at Station R (September 1981). Figure 3 shows that the rate of Trp destruction (and thus the PSE) increases with the DOM content of a sample. The aquarium water, which is enriched with metabolic products of the fauna and flora, is much more efficient photosensitizing media than seawater. In Figs. 5, 6, 7 and 8 the variations of the temperature and the PSE of seawater samples taken at various depths in several stations, are shown. Station A 1

From the top towards the bottom, the temperature profile indicates that the upper layer {ca. 10m thickness) is slowly warming (16.2--15.4°C) at thiz time of the year. A thermocline formation is under way between 10m and 30m followed by cooler waters (13.6°C). Variance analysis on the PSE values obtained with the upper layer (0--10m) versus those found in the (20--100 m) layer indicates significant differences (1% < F < 5%) (where F is the Fisher's variance ratio in analysis of variance) between the PSE obtained with unfiltered water in the upper (average 10.22) and in the lower layers (average 5.88). Figure 5 suggests that the PSE change occurs at the thermocline level. On the other hand with filtered water, one observed a random PSE distribution (average 6.06). Station A 2

The temperature profile at the same station at the end of summer indicates a pronounced stratification of the water layers as shown by the thermocline (21.1°C--15.1°C).

]2

Regression calculation carried out on the PSE obtained with unfiltered water shows an excellent correlation (R :: 0.882) (Fig. 6) between 0.1m and 500m according to the function y =- ae bx (a = 8.125, b := - 0 , 0 0 4 9 1 ) , where x is the PSE value. With filtered water a good correlation (R - 0.554) is observed according to the function y - a + b In x provided one ignores the unexpected result obtained at 150m. Taking this value into account leads to R -- 0.459 using the function y = a x h . In conclusion, the PSE of samples collected at Station A either in spring or at the end of the summer notably decreases with depth, at least for unfiltered samples, as shown by statistical analysis. From 0.1 m to 50m there are no significant differences in the PSE of filtered and unfiltered waters. The difference between the PSE of filtered and unfiltered water at depth ~ 100m may reflect the effect of particles in unfiltered water either on the absorption of the DOM or their interference with fluorescence measurements. The strong variation of PSE from the air--water interface through the euphotic layer is further exemplified by results obtained at Station C (Fig. 7) where there is a sharp drop of the PSE within a few decimeters. In contrast with Station A, results obtained at Station C reflect samples devoid of any characterized anthropogenic pollution. It must be noted that the samples at Station C as well as those at Station A~ have been collected under very favorable sea conditions. On the other hand, the random distribution of PSE values determined at Station R {Fig. 8) corresponds to samples collected under rough conditions leading to perturbation of the water column. CONCLUSIONS

It must be stressed that the PSE values obtained near the air--water interface in fair sea conditions are rather high (average: Station A1 = 10.22, Station A2 = 9.73 and Station C = 9.03}. The average very low concentration of riboflavin and pteridine derivatives f o u n d in the Mediterranean sea at the time of the collection amounts to ~ 10-10 M (for instance at Station A~ : RF ~ 0.04pg/1, pterins ~ 0 . 0 1 ~ g / l ) and cannot therefore account for the observed PSE. Consequently, other photosensitizers such as chlorophyll derivatives, humic and fulvic acids, bile-type pigments etc., must be present in reasonable amounts. This has to be clearly established, especially in areas where conditions are nearly ideal with respect to the absence of pollution since the presence of these natural photosensitizers would automatically induce an abiotic transformation of the DOM. Moreover, these photosensitized degradations will be maximal in the air--sea interface film where the substrate concentrations are much higher than in the rest of the sea. In this respect, the presence of unsaturated f a t t y acids susceptible to photooxidation via radical chain reactions may play a key role by forming 'reaction centers' where organic substances of the DOM could be modified or synthesized via radical chemistry.

13 ACKNOWLEDGEMENTS

The authors are pleased to acknowledge the skillful assistance of Mrs. G. Chennebault~:~ondry and of the crew of the O/S. Winnaretta-Singer. The collection of seawater samples has been made possible through the permission of Cdt. J.Y. Cousteau, Director of the Mus6e Oc6anographique at Monaco. This work has been granted by the Centre National de la Recherche Scientifique through contract ATP-3491, Oceanographie Chimique. REFERENCES Albert, A. and Wood, H.C.S., 1953. Pteridin syntheses II. J. Appl. Chem., 3: 521--523. Aubry, J.M., Rigaudy, J. and N.K. Cuong, 1981. Kinetic studies of self-sensitized photooxygenation in H20 and D20 of a water-soluble rubrene derivative. Photochem. Photobiol., 33: 155--158. Borg, D.C., Schaich, K.M., Elmore, J.J., Jr. and Bell, J.A., 1978. Cytotoxic reactions of free radicals species of oxygen. Photochem. Photobiol., 28: 887--907. Bourdon, J., 1972. Effet photodynamique et oxydation photosensibilis~e. Applications aux enzymes. In: Ecole de Roscoff: La photobiologie, Edition du C.N.R.S., Paris, pp. 67--85. Chahidi, C., Aubailly, M., Momzikoff, A., Bazin, M. and Santus, R., 1981. Photophysical and photosensitizing properties of 2-amino-4-pteridinone, a natural pigment, Photochem. Photobiol., 33: 641---649. Escande, C., Bousquet, B. and Dreux, C., 1977. Dosage fluorim~trique du tryptophane libre et total dans le plasma sanguin. Ann. Biol. Clin., 35: 387--395. Foote, C.S., 1976. Photosensitized oxidation and singlet oxygen: consequences in biological systems. In: W.A. Pryor (Editor), Free Radicals in Biology. Academic Press, New York, pp. 85--133. Garrett, W.D., 1965. Collection of slick-forming materials from the sea surface. Lhnnol. Oceanogr., 10: 602--605. Gollnick, K., 1975. Chemical aspects of photodynamic action in the presence of molecular oxygen. In: O.F. Nygaard, H.I. Adler and W.K. Sinclair (Editors), Radiation Research: Biomedical, Chemical and Physical perspectives. Academic Press, New York., pp. 590--611. Joussot-Dubien, J. and Kadiri, A., 1970. Photosensitized oxidation of ammonia by singlet oxygen in aqueous solutions and seawater. Nature, 227 : 700--701. Landymore, A.F. and Antia, N.S., 1978. White light promoted degradation of Leucopterin and related pteridines dissolved in seawater with evidence for involvement of complexation from major divalent cations of seawater. Mar. Chem., 6 : 309--325. Lyman, J. and Fleming, R.H., 1940. Composition of seawater. J. Mar. Res., 3: 134--146. Momzikoff, A., 1977. Substances fluorescentes (pt~rines et flavines) dans les eaux de mer et planctons matins. Essai d'interpr~tation ~cologique. Thesis, Paris. Momzikoff, A. and Santus, R., 1981. Sur les propri~t~s photosensibilisatrices des pt~rines. Exemple de la biopt~rine. Comparisons avec la riboflavine. C.R. Acad. Sci. Set. C, 293: 15--18. Ogflby, P.R. and Foote, C.S., 1981. Unexpected solvent deuterium isotope effects on the lifetime of singlet molecular oxygen (1 ~ ) . j. Am. Chem. Soc., 103: 1219--1221. Parker, C.A., 1968. In: Photoluminescence of Solutions. Elsevier, Amsterdam, pp. 208-214. Pileni, M.P., Santus, R. and Land, E.J., 1978. On the photosensitizing properties of N-formylkynurenine and related compounds. Photochem. Photobiol., 28: 525--529. Rio, G. and Scholl, M.J., 1975. Photochemistry of 1, 3 diphenyl isobenzofuran. J. Chem. Soc. Chem. Commun., 474--476.

14 Walrant, P. and Santus, R., 1974. N-formylkynurenine, a tryptophan photo-oxidation product as a photodynamic sensitizer. Photochem. Photobiol., 19: 411--417. Walrant, P., 1976. Contribution h l'~tude des propri~t4s photosensibilisatrices d'un produit de photo-oxidation du tryptophane: la N-formylcynurenine. Thesis, Orsay, Paris. Wolff, C.J.M., Halmans, M.T.H. and van der Heijde, H.B., 1981. The formation of singlet oxygen in surface waters. Chemosphere, 10: 59--62. Zafiriou, O.C., 1977. Marine organic photochemistry previewed. Mar. Chem., 5 : 497--522. Zika, R.G., 1981. Marine organic photochemistry. In: E.K. Duursma and R. Dawson (Editors), Marine Organic Chemistry. Else~er, Amsterdam, pp. 299--325. Zepp, R.G., Wolfe, N.L., Baughman, G.L. and Hollis, R.C., 1977. Singlet oxygen in natural waters. Nature, 267: 421--423. Zepp, R.G., Baughman, G.L. and Schlotzhauer, P.F., 1981. Comparison of photochemical behaviour of various humic substances in water. I. Sunlight induced reactions of aquatic pollutants photosensitized by humie substances. Chemosphere, 10: 109--117.