Photogeneration of singlet oxygen by humic substances:comparison of humic substances of aquatic and terrestrial origin

Andrea Paul,*a Steffen Hackbarth,b Rolf D. Vogt,c Beate Röder,b B. Kent Burnison d andChristian E. W. Steinberg a

a Leibniz Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 301,D-12561 Berlin, Germany

b Humboldt University Berlin, Institute for Physics, Newtonstr. 15, D-12489 Berlin, Germanyc Department of Chemistry, University of Oslo, N-0315 Oslo, Norwayd Environment Canada, National Water Research Institute, 867 Lakeshore Road, Burlington,

Ontario L7R 4A6, Canada

Received 1st October 2003, Accepted 1st December 2003First published as an Advance Article on the web 9th January 2004

The singlet oxygen (1O2) luminescence of 27 isolated humic substances (HS), natural organic matter, ultrafiltrates,and the synthetic fulvic acid HS1500 has been investigated by time-resolved spectroscopy in buffered D2O. Thesamples include both reverse osmosis isolates from lakes in Scandinavia, Canada, and Germany, and IHSS fulvic andhumic acids of aquatic and terrestrial origin. The quantum yields of 1O2 formation (Φ∆) obtained on laser excitationat 480 nm ranged between 0.06 (HS1500) and 2.7% (fulvic acid from soil, IHSS). In our study, a general trendtowards higher Φ∆ in terrestrial HS was observed. The comparison of reverse osmosis isolates from surface waterscollected during fall 1999 and spring 2000 from five Scandinavian sites yielded, in all cases, higher Φ∆ for the springsamples. For the aquatic sampling sites Hietajärvi and Birkenes, Φ∆ even exceeded values of 0.6%, which were foundto be typical for terrestrial or soil water material. Investigation of the excitation wavelength dependence of Φ∆ in thespectral range 355–550 nm yielded different spectral shapes for aquatic HS and “non-aquatic” HS, respectively. Onthe basis of these excitation spectra, 1O2 production rates were calculated for eight representative HS.

IntroductionIn natural waters, humic substances (HS) represent the mainfraction of dissolved organic carbon that absorb solar radiationand, therefore, play an important role in aquatic photo-chemistry.1 Photolysis of HS leads to the formation of a com-plex mixture of reactive species, including free radicals andhydrated electrons,2 and reactive oxygen species such as singletoxygen (1O2),

3–6 hydroxyl radicals,5,7,8 superoxide,5,9 hydrogenperoxide,5,10,11 and reactive triplet states.12,13 In addition, smallerfragments (e.g. short chain fatty acids, aldehydes,14,15 and aminoacids 16) are released. During these processes, the HS undergophototransformations, including partial degradation,17,18 andphotomineralization of HS may occur.19,20 As a result of thesephotoreactions, both beneficial and adverse effects on aquaticorganisms are reported. The fatty acids, carbonyl compounds,and amino acids which are released serve as organic nutrientsfor heterotrophic bacteria,21,22 while long-lived reactive speciesare, in some cases, reported to have adverse effects on aquaticorganisms.23–25 These seemingly contradictory results demon-strate the ecological relevance of a detailed photochemical andphotophysical investigation of HS.

1O2 is considered to be the most reactive oxygen speciesgenerated upon illumination of the majority of organic dyesin aerobic solution.26 In aquatic ecosystems, 1O2 is argued to beresponsible for the indirect photochemical degradation ormodification of organic pollutants,27 such as phenols,28,29 andallelochemicals, such as cyanotoxins.30,31 Furthermore, 1O2 is anefficient oxidant for a variety of unsaturated organic com-pounds that is able to oxidize membranes, proteins, and nucleicacids.32,33 The formation of 1O2 is achieved by energy transferfrom the first excited triplet state of the dye molecules, which ispopulated following light absorption and intersystem crossing.1O2 then interacts with other molecules by energy transfer andchemical reactions, including interactions with the sensitizeritself or formation of charge-transfer intermediates that can

lead to superoxide radicals.1,34 The typical phosphorescenceof 1O2 at 1269 nm can be used to calculate quantum yields of1O2 formation (Φ∆). The radiationless route of singlet oxygenphysical deactivation is solvent dependent. Φ∆ can thereforebe determined either by steady-state or time-resolved methods.Lifetimes of 3.1 and 62 µs are found for 1O2 in H2O and D2O,respectively.35

Here, we present novel time-resolved measurements of 27natural HS and one synthetic fulvic acid (HS1500) in bufferedD2O. The samples include reverse osmosis (RO) isolates fromScandinavian sites taken in different seasons (fall 1999 andspring 2000), RO from USA and Germany, ultrafiltrationisolates (‘NOM’) from Canada, Laurentian fulvic and humicacids, and IHSS fulvic and humic acids from the SuwanneeRiver, Florida peat and Summit Hill soil, Elliot soil and Leon-ardite HS. The focus of this paper is the comparison of (i)terrestrial, aquatic, coal, and synthetic HS and NOM and (ii)seasonal variations of the quantum yield of 1O2 formationupon excitation at 480 nm. In addition, excitation wavelength-dependent quantum yields of 1O2 formation, which enablethe calculation of action spectra and 1O2 sensitization rates,were determined in the spectral range 355–550 nm for eightrepresentative HS.

Materials and methods

Humic substances

The samples included one synthetic fulvic acid (HS1500 36,37),various humic acids (HA), and fulvic acids (FA), and severalRO isolates of natural water samples (“natural organic matter”,NOM), and ultrafiltrate isolates. The HS1500 was derived fromautooxidation of hydroquinone and has a molecular mass ofabout 1500 Daltons. Laurentian FA and HA from Laurentiansoil were purchased from Fredriks Research Products.38 HAand FA from Elliott soil, HA from Leonardite, Summit HillD

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and Pahokee peat, and HA, FA, and NOM from the SuwanneeRiver were acquired from the International Humic SubstancesSociety (IHSS).39 The Suwannee River is a blackwater river thatrises in the Okefenokee Swamp in southern Georgia. The Elliottsoil is typical of the fertile prairie soils of the American statesof Indiana, Illinois, and Iowa. The Leonardite HA is producedby the natural oxidation of exposed lignite, a low grade coal,from the Gascoyne Mine in Bowman County, North Dakota.The Summit Hill soil {Orthic Brown Soil (NZ); TypicDystrudept (USDA)} is taken from a tussock grass area onthe South Island of New Zealand. The Pahokee peat is atypical agricultural peat soil from the Florida Everglades. ROisolates of NOM were received from the NOMiNiC (NaturalOrganic Matter in the Nordic countries) project 40 and from theLeibniz Institute of Freshwater Ecology and Inland Fisheries(IGB). Detailed characterizations of the Scandinavian ROisolates from Birkenes, Hiatajärvi, Skjervatjern, Svartberget,and Valkea-Kotinen are given by Vogt et al.,41 and forHellerudmyra by Gjessing et al.42 Briefly, the Hietajärvisite comprises a large oligotrophic lake, the Skjervatjernsite is a small dystrophic lake, and Lake Valkea-Kotinen is asmall humic and acidic headwater lake. Both the Birkenesand Svartberget sites are small streams. Svartberget is a borealpristine one, while Birkenes lies in the southern part of Scandi-navia that received the highest anthropogenic S-deposition. TheFuchskuhle NOM was isolated by the IGB from the south-western basin of the dystrophic Lake Fuchskuhle in Germany 43

according to the procedure described by the IHSS.44 Ultra-filtration was applied to surface waters of Luther Marsh,Beverly Swamp, and Sanctuary Pond near Ontario, Canada.45

To differentiate between the RO and the ultrafiltrate naturalorganic matter, the latter are denoted ‘NOM’, in invertedcommas. The results of the elemental analyses of the GermanNOM from Lake Fuchskuhle and the Canadian ‘NOMs’from Luther Marsh, Beverly Swamp, and Sanctuary Pond aresummarized in Table 1. All the HS investigated here, togetherwith its abbreviation, source, and ecological origin are listed inTable 2.

Chemicals

D2O, Rose Bengal (fluorescein 3,4,5,7-tetrachloro-2�,4�,5�,7�-tetraiodo dianion), NaN3, and buffer salts (anhydrous sodiumhydrogen phosphate and sodium dihydrogen phosphate) werepurchased from Aldrich and Merck, respectively.

Time-resolved determination of singlet oxygen quantum yield anddecay times

The experimental set up has been described in detail previouslyby Oelckers et al.46 Briefly, excitation of samples was performedusing a Nd:YAG laser-pumped BMI optical parametric oscil-lator (OPO) and a 5 × 5 mm2 Ge-Pin diode with cooled pre-amplifier (Northcoast) was used for detection. To achieve thehighest signal to noise ratio, the sample was positioned directlybefore the diode, separated only by long pass and metalinterference filters. The signal measured was the typical weakphosphorescence of singlet oxygen at 1269 nm. The signal wasisolated from scattering and fluorescence by the different time-scales of the processes.

In case of short triplet decay times of the sensitizer, themeasured decay curves can be fitted mono-exponentially with aleast-squares method, yielding the value for the initial ampli-tude and the decay time. The amplitude is directly proportionalto the amount of singlet oxygen generated, whereas the decaytime gives information about the quenching processes of singletoxygen. Under the precondition that both sensitizer and refer-ence are adjusted to the same optical density at the excitationwavelength, singlet oxygen quantum yields, Φ∆, can be calcu-lated by comparing the amplitudes of the samples, I, with thatof the reference, I ref, (eqn. 1). T

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Table 2 Singlet oxygen quantum yields (Φ∆) and 1O2 decay times (τP) for natural and synthetic HS and NOM in D2O

Sample Abbreviation Source Origin Φ∆ (%) τP/µs

Valkea-Kotinen NOM (fall 1999) Val-f NOMiNiC Aquatic 0.16 66Birkenes NOM (fall 1999) Bir-f NOMiNiC Aquatic 0.19 55Luther Marsh ‘NOM’ Lu-‘NOM’ Burnison Aquatic 0.21 48Skjervatjern NOM (fall 1999) SKJ-f NOMiNiC Aquatic 0.22 48Suwannee River NOM SuRi-NOM IHSS Aquatic 0.23 55Svartberget NOM (fall 1999) SVA-f NOMiNiC Aquatic 0.26 51Nordic reference NOM NOR-NOM Gjessing Aquatic 0.29 50Skjervatjern NOM (spring 2000) SKJ-s NOMiNiC Aquatic 0.29 66Valkea-Kotinen NOM (spring 2000) Val-s NOMiNiC Aquatic 0.30 61Svartberget NOM (spring 2000) SVA-s NOMiNiC Aquatic 0.30 51Suwannee River HA standard SuRi-HA IHSS Aquatic 0.31 43Sanctuary Pond ‘NOM’ Sa-‘NOM’ Burnison Aquatic 0.38 51Hellerudmyra NOM Heo-NOM Gjessing Aquatic 0.43 58Fuchskuhle NOM Fuku-NOM IGB Aquatic 0.48 44Suwannee River FA standard SuRi-FA IHSS Aquatic 0.57 42Hietajärvi NOM (fall 1999) Hiet-f NOMiNiC Aquatic 0.59 47Hietajärvi NOM (spring 2000) Hiet-s NOMiNiC Aquatic 0.86 46Birkenes NOM (spring 2000) Bir-s NOMiNiC Aquatic 0.91 47Beverly Swamp ‘NOM’ Bev-‘NOM’ Burnison Swamp 0.61 46Peat HA reference Peat(R)-HA IHSS Peat 0.81 58Peat HA standard Peat(S)-HA IHSS Peat 0.83 61Laurentian FA Lau-FA Fredriks Terrestrial 0.26 57Leonardit HA standard Leon-HA IHSS Terrestrial 0.55 53Summit Hill Soil HA reference Sum-HA IHSS Terrestrial 0.67 58Soil HA standard Soil-HAII IHSS Terrestrial 0.75 60Laurentian HA Lau-HA Fredriks Terrestrial 1.08 53Soil FA standard II Soil-FAII IHSS Terrestrial 2.69 58HS1500 HS1500 Weyl GmbH Synthetic ≤0.06 n.d.

For the available excitation range (355–550 nm), Rose Bengal(Φref

∆ = 75% in D2O47) was used as a reference sensitizer.

Absorption coefficients

Absorption coefficients of HS in buffered D2O (pH ∼7.5) werecalculated on the basis of absorption spectra measured using anUvicon spectrometer.

Results

Singlet oxygen measurement

A typical time-resolved emission for 1O2 derived from HS inbuffered D2O (pH approx. 7.5) is presented in Fig. 1(A). Usingthe temporal region between 25 and 200 µs for fitting, in allcases, single-exponential fits were obtained. In the temporalrange below 20 µs, a so-called scattering peak and a short-livedcomponent were observed. Despite the fact that two filters wereused to reduce signals due to both scattering and fluorescence,the short-lived signal can be several orders of magnitude higherthan the 1O2 luminescence. Therefore, detector artefacts thatmay occur at longer timescales have to be excluded. These arte-facts depend mainly on the intensity of the short-lived signaland can be visualized by adding 40 mM NaN3 to the sample.The remaining signal, which is then proportional to the decayof the sensitizer triplet state, can be neglected. Because of thehigh concentration of NaN3, the intensity of 1O2 luminescenceis reduced to values smaller then the noise of the detector signaldue to dynamic quenching.34 Hence, the influence of thedetector artefacts can be reduced by subtracting the detectorsignal of the same sample containing NaN3. Obviously, themain influence of the detector artefacts is on a timescale of lessthan 20 µs. Therefore, the long decay time of 1O2 in D2O enablesus to perform a fit outside of this timescale and, thereby, toachieve reliable results [Fig. 1(B)]. The values determined repre-sent the average of 2 to 6 single measurements and have arelative accuracy of about 12.5%.

(1)Comparison of �� for HS of different origins

In Table 2, both decay times (τP) and quantum yields (Φ∆) of1O2 are summarized. All samples were adjusted to an OD of0.2 at the excitation wavelength (480 nm). Consequently, theobtained τP reflect the dilution of the HS samples, which differsaccording to the absorption coefficients, rather than thequenching behavior. The values of Φ∆ vary by more than oneorder of magnitude. The lowest Φ∆ was found for the syntheticHS1500 (Φ∆ ≤ 0.06%, due to the weak signal, no proper fitcould be performed). This result is in agreement with the find-ings of Aguer and Richard,48 who could not detect any 1O2

formation with a synthetic HS by using the trapping method.Low quantum yields were also obtained for several RO isolatesand SuRi-HA. Intermediate Φ∆ values were measured withSuRi-FA, Lau-FA, and several NOM and ‘NOM’. Except forthe spring RO isolates from Birkenes and Hietajärvi, highervalues (Φ∆ > 0.6) were generally obtained with HA derived fromsoil, coal, and peat (Fig. 2). A comparison between NOM, HA,and FA for the SuRi-samples yielded Φ∆ values of 0.23, 0.31,and 0.57%, respectively. The same pattern emerged for soil-HAand soil-FA, with Φ∆ = 0.75 and 2.7%, respectively, whereas theLaurentian HA and FA behave in the opposite manner, with Φ∆

= 1.1 and 0.26%, respectively. The trend of higher Φ∆ for FAthan HA confirms the results of Sandvik et al.,49 who found thehighest Φ∆ occurred for the lower molecular mass material insize-fractionated NOM. Among all HS investigated here, theIHSS soil-FAII, gave a particularly high Φ∆. In general, the FAfraction is characterized by lower molecular weight, lower levelsof total and aromatic carbon and a greater proportion of oxy-gen-containing functional groups than the HA fraction.50 Ourresults indicate either a higher concentration of the sensitizingcompounds in the FA fraction or less quenching of the excitedstates of these compounds. It is not possible to distinguishwhich of these possibilities is applicable; therefore, furtherexperiments are planned to investigate the quenching behaviorof HS.

The comparison of HS from different origins (surface waters,peat, soil, coal) in Fig. 2 indicates, in general, lower Φ∆ valuesfor those HS which were derived from surface waters and higherΦ∆ for those which were derived from terrestrial ecosystems or

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peat. Nevertheless, three exceptions were observed: the soil HSLau-FA, which shows an untypically low Φ∆, and the aquaticHS Bir-s and Hiet-s, which show untypically high Φ∆ values. Incontrast to the other HS, which were purchased by the IHSSor donated by groups working with HS (cf. Materials andmethods), Lau-FA and Lau-HA were the only commerciallyavailable HS and no details of their preparation were available.Therefore, it cannot be excluded that the different treatment ofsamples during separation and purification of the LaurentianHS is the reason for the non-typical data. A possible explan-ation for the comparable high Φ∆ values for Hiet-s and Bir-s,which were both collected in spring during the NOMiNiCproject, will be given in the following section.

Seasonal variations of NOM samples from the NOMiNiCproject

Among the aquatic samples that are mainly characterized bylow and medium Φ∆, some NOM exhibit Φ∆ values comparableto those for the NOM from terrestrial samples or peat (Table 2).The direct comparison of samples from fall and spring pre-sented in Fig. 3 reveals higher Φ∆ values for the spring samplesin all cases; the largest differences between spring and fall werefound for Birkenes and Hietajärvi. As part of the NOMiNiCproject,40 correlations between site characteristics and severalstructural and chemical parameters of all 10 HS were per-formed.41 Based on differences in the catchment characteristics,

Fig. 1 (A) Time-resolved phosphorescence of 1O2 from Peat(S)-HAin D2O: (1) experimental trace; (2) single-exponential fit of trace 1,(3) experimental trace after addition of 40 mM NaN3. Excitationwavelength 480 nm, OD(480 nm) = 0.2, emission wavelength 1269 nm.(B) Time-resolved phosphorescence and corresponding mono-exponential fits of representative HS in the temporal range 25–195 µs.Excitation wavelength 480 nm: (1) Soil-FAII; (2) Peat(S)-HA; (3) Bir-s;(4) Bir-f; (5) SuRiHA; (6) Val-s; (7) phosphorescence quenched byNaN3.

it is not clear why the observed effect is so pronounced inthese samples. As was mentioned in the Materials and methodssection, Hietajärvi is a lake and Birkenes a small stream. On theother hand, the Φ∆ values among the other samples was foundto correlate well with a number of NOM properties: FT-IRabsorption between 1000–1060 cm�1 (r = 0.818), specificvisual absorption (sVISa; r = �0.699), specific absorption ratio(SAR; r = 0.709), hydrophilic neutrals (HPI-N; r = 0.753) andnitrate concentration in the re-dissolved RO solution (r =0.875). The IR absorption between 1000–1060 cm�1 in HS canbe assigned to polysaccharides and methoxy groups,51 sVISa isdefined as the quotient Aλ400nm/mg(C) L�1 and SAR representsthe quotient Aλ254nm/Aλ400nm. High SAR is expected for materialwith low molecular weight,52 but does not correspond to thelevel of condensed aromatic rings.53 In addition, the SAR isexpected to increase with increasing oxygen content.54 Whereasthe correlation with nitrate concentration is not understood atpresent, the other correlations point to colored, oxygen-richstructures with low molecular weight which are mainly involvedin 1O2 production. This characteristic agrees with the descrip-tion of fulvic acids 50 given above.

The seasonal variation of Φ∆ indicates that modificationsof the photoreactivity occur during both summer and winter.It can be speculated that during fall, winter, and spring,processes like degradation of litter by microorganisms and anincreased input of terrestrial material as a result of extensiverainfall may contribute HS structures to surface waters, which,in turn, may give rise to the formation of ‘fresh’ and morephoto-reactive HS. During summer time, photochemicalmodification of NOM may instead be the dominant process,where continuous photomineralization results in the des-truction of those structures that are responsible for 1O2

sensitization.

Fig. 2 Singlet oxygen quantum yields (Φ∆, mean ± accuracy 12.5%)upon excitation at 480 nm of aquatic HS ( ), HS derived from peat orswamp (�), and terrestrial HS (�) in buffered D2O, sorted according tosample number in Table 2.

Fig. 3 Seasonal variation (grey: fall 1999; black: spring 2000) in Φ∆

(mean ± accuracy 12.5%) for five Scandinavian sampling sites.

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Fig. 4 (A) Wavelength dependence of Φ∆ (mean ± accuracy 12.5%) from excitation spectra of HS. (B) Action spectra of HS samples in D2O. (C) 1O2

production rates for HS in water calculated for the surface according to Haag et al.4 and Zepp and Cline.55 (D) 1O2 production rates for HS in watercalculated for the whole water body using the W values from Zepp and Cline 55 for midday, summer sunlight, latitude 40� N. HS: (�) Hiet-f; (�) SuRi-NOM; (�) SuRi-HA; (�) Sva-s; (�) Peat(S)-HA; (�) Bir-s; ( ) Bir-f; ( ) Soil-FAII.

Dependence of ��on excitation wavelength

The wavelength-dependent quantum yields of 1O2 formation[excitation spectra, Fig. 4(A)] were determined for the samplesSuRi-HA, SuRi-NOM, Peat(S)-HA, Bir-f, Bir-s, Soil-FAII,Hiet-f, and Svart-s. For each excitation wavelength, a newsample was prepared and adjusted to an OD of 0.20. The oper-ation mode of the Nd:YAG laser system working with a SHGcrystal plus an OPO enabled a tuning range between 450–550nm, and without OPO excitation at 355 nm. Due to the fact thatHS represent a complex mixture of various molecules, the con-tribution of the absorption of the 1O2 generating species tothe overall absorption of HS varies with wavelength and, con-sequently, a dependence of Φ∆ on the excitation wavelength canbe expected. All the aquatic NOM samples yield a similarspectral shape [Fig. 4(A)], which is characterized by an increasefrom 550 to 450 nm and a weaker slope from 450 to 355 nm.There could be a maximum around 450 nm, but, because of thenon-accessible excitation range between 450 and 355 nm, themaximum also could be blue shifted. Nevertheless, we postulatethat similar chromophores or chromophoric systems areresponsible for the photosensitized formation of 1O2 in theseHS. Interestingly, HS from peat (Peat-S) and soil (soil-FAII)exhibit a similar spectral shape in the range 450–355 nm. Incontrast to the aquatic samples, no further increase in Φ∆ at 355nm was found and the presence of a maximum at ca. 450 nm ismore evident. Soil-FAII exhibits an extraordinarily high Φ∆

over the whole spectral range. The lowest Φ∆ values were foundfor SuRi-HA, which exhibits an intermediate spectral shape.Our results indicate that there are two main groups of chromo-phores in HS which contribute to 1O2 formation: one groupabsorbing in the spectral range 355–550 nm with a maximumaround 450 nm, which is present in both fractionated HS and inNOM, and a second group of chromophores which is respon-sible for the increase in Φ∆ in the UV range, dominating the

excitation spectra of aquatic NOM. The latter chromophoresare either not contained in soil/peat HS or they were removedor destroyed during the process of HA/FA fractionation.

The product of the mass-based wavelength-dependentabsorption coefficients [ε(λ), cf. Fig. 5] with Φ∆ provides theaction spectra for 1O2 production.4 The action spectra inFig. 4(B) are clearly dominated by the value of ε(λ). Peat-HAexhibits highest values, followed by soil-FAII, whereas compar-able low values are obtained for all aquatic NOM.

Our calculation of singlet oxygen production rates (d[1O2]/dt)is based on the underwater irradiation intensities of Zepp andCline,55 in order to compare our results with the data presentedby Haag et al.4 The amount of singlet oxygen generated isgiven by the product of the number of absorbed photons

Fig. 5 Underwater irradiation intensities according to Zepp andCline,55 W values (1), Z values (2), and absorption coefficients (ε) for HSin the spectral range 300–800 nm. HS with increasing ε: Bir-f, Bir-s,Hiet-f, SuRi-NOM, SuRi-HA, Soil-FAII, Svart-s, Peat-S-HA.

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Table 3 1O2 production rates for representative HS samples in D2O (pH ∼7.5) calculated according to Haag et al.4 based on the irradiation data ofZepp and Cline 55 for summer sunlight at latitude 40� N at midday

Sample (d[1O2]/dt)/10�11 mol cm�3 s�1 (d[1O2]/dt)/10�9 mol cm�2 s�1

Bir-f 0.16 0.94Bir-s 0.12 1.30Sva-s 0.34 0.42Hiet-f 0.16 0.59SuRi-NOM 0.23 0.66SuRi-HA 0.28 0.40Peat(S)-HA 4.77 1.10Soil-FAII 1.84 3.43

and the wavelength-dependent singlet oxygen quantum yield(Φ∆λ, cf. eqn. 2),

where IHS is the number of photons absorbed by the humicsubstance. Depending on the optical density of the waterunder investigation, solar irradiation will be attenuated to adifferent degree and, hence, two major cases can be dis-tinguished: (i) shallow depth in any natural water with overallOD < 0.1 and (ii) higher penetration depth or deeply coloredwater with overall OD > 2.0. In general, eqn. 3 describes thelight intensities absorbed by HS,

where D is the depth, ε is the mass-based absorption coefficientof HS, c is the concentration of HS, α is the light attenuationcoefficient of pure water (< 0.001 m�1), and Id and Is are theirradiation intensities due to direct sunlight and sky irradiation,respectively. ld and ls are the pathways in the water body to adepth D.55 IHS is proportional to its relative contribution to thelight attenuation in the water body. Considering a concen-tration of 1 mg L�1 HS in a volume of 1 cm2 × D [case (i)], theintensity can be estimated to be constant in the whole waterbody. This allows for an approximation of eqn. 3 with lineardependency,

where Z is the angle-corrected irradiation for 40� N sunlight atmidday (Id × ld � Is × ls)

55 and f is the correction factor toobtain a linear approximation. The error caused by this approx-imation is less than 6% for an overall OD < 0.1 if we choose f tobe 2.17. The wavelength-dependent 1O2 production rates for theHS under investigation for a depth of only 1 cm are presentedin Fig. 4(C). It is obvious that Φ∆λ, ε(λ) and Z(λ) mainly deter-mine the spectral distribution of the rate of generated 1O2. Dueto the spectral shape of sunlight (Z, cf. Fig. 5), the achievedmaximum 1O2 generation lies in the visible range. Integrationover the whole spectral range yields the 1O2 production rates inTable 3.

In the case of greater depths or high HS concentrations,nearly all incoming light is absorbed by the HS in the waterbody, because even for HS with a low extinction coefficientand a concentration of only 1 mg L�1, the attenuation due tothe water is about 5 times smaller than the attenuation dueto the HS. Consequently, IHS equals the overall illumination(W values,55 cf. Fig. 5). Therefore, the overall production rateis only dependent on the illuminated surface of the incidentlight, rather than on the volume. Here, only the spectrum ofsolar irradiation and Φ∆λ determine the spectral shape of 1O2

production rates [Fig. 4(D), Table 3].

(2)

(3)

IHS = Zfcε(λ)D (4)

DiscussionOur investigations revealed that the characteristic phos-phorescence of 1O2 at 1270 nm of photoexcited HS can bemeasured by time-resolved optical methods. In previousstudies, chemical methods that permit the accumulation ofphotoproducts have been applied.1,4,5 The chemical detection ofsinglet oxygen by using furans as traps has evidenced thatfurans can sometimes lack specificity.34 With the developmentof a highly sensitive experimental set-up, it is feasible to per-form optical investigations of quantum yields in scatteringsamples.46 In principle, singlet oxygen luminescence can beinvestigated both by steady-state and time-resolved measure-ments. Steady-state measurements as performed by Sandviket al.49 are very sensitive to variations in the decay time of 1O2

because the measured intensity is proportional to the productof amplitude and decay time. Hence, a reduction in the lifetimedue to quenching processes leads to smaller values of Φ∆

determined this way. In contrast to steady-state measurements,time-resolved investigations need no correction because thisinformation is included in the decay curve. Here, we presentnovel (to the best of our knowledge) singlet oxygen quantumyields of HS determined by time-resolved phosphorescencemeasurements.

The investigation of Φ∆ upon excitation at 480 nm indicated aclear trend of higher Φ∆ for terrestrial HS and HS derived frompeat than for most of the aquatic HS. Those aquatic NOMsthat were collected in springtime exhibited higher Φ∆ in all casesthan samples from the same catchment collected in the fall.Looking at the 1O2 action spectra and formation rates, the trendof high Φ∆ for ‘non-aquatic’ HS is even more pronounced. Theresults in Fig. 4(B)–(D) and Table 3 clearly show that over thewhole spectral range investigated, ‘non-aquatic’ samples mustbe considered to be more photoreactive. Nonetheless, majordifferences between the 1O2 production rates were observedwhen only the top layer of natural waters [D = 1 cm, Fig. 4(C)]or a deeper water body [Fig. 4(D)] were investigated. It can beexpected that at the surface of natural waters, HS which arecharacterized by high ε(λ) and high Φ∆ will produce greatestamounts of 1O2. At 1 cm depth, UV irradiation will contributeto the observed overall production of 1O2 and ‘non-aquatic’HS will produce the highest 1O2 rates. In contrast, consideringa lake with a depth of some meters and/or with highly coloredwater, the differences between the 1O2 production rates ofaquatic and ‘non-aquatic’ HS become less pronounced. Ourresults for aquatic HS in the surface layer can be compared withthose of Haag et al.,4 for blackwater NOM and Lake BaldeggNOM (cf. Fig. 6) in a similar spectral range (366–546 nm). BothΦ∆λ and 1O2 production rates for ‘non-aquatic’ HS exceed thevalues presented by Haag et al.4 However, there was no corre-lation to the Φ∆ values measured by Shao 56 and reported byScully et al.57 for three natural waters in the spectral range 300–400 nm (Fig. 5), which seem comparably low. Literature valuesfor Φ∆ exciting between 300 and 400 nm generally rangebetween 1 and 3%.3–5 In one case, a value of 6% was found,49

which is in good agreement with the Φ∆366nm (1–3.7%) andΦ∆480nm (0.6–5.5%) values obtained in our study.

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Our data demonstrate that HS are by no means recalcitrant,or even inert, molecules that remain stable over long periods. Inaddition to a large spatial variation, a temporal variation ofHS with respect to its photoreactivity has to be taken intoaccount. Considering the ecological relevance of the results ofour investigations, several questions arise. What happens whenHS of terrestrial origin are transferred into lakes and rivers?Which processes play a dominant role in the transformation ofmore reactive terrestrial matter into relatively less reactivematter?

Aquatic HS exhibit characteristics that are probably due tophotolysis, but nonetheless, aquatic HS originates in terrestrialareas. Therefore, they also reflect the impact of turnover anddeterioration processes in surface soils or peat. In soils andpeat, HS become photolysed only on the surface, whereasin aquatic systems, HS are exposed as long as they remain inthe photic water layer. As a consequence, aquatic samplescollected in spring suffer less from the impact of solar irradi-ation than samples collected in the fall. Spring samples displayhigher Φ∆ than fall samples because of processes such as photo-mineralization and photomodification of HS structures. Ourdata suggest that reactive oxygen species are more abundantin the spring than in the fall, indicating that either potentially1O2-producing NOM structures in older humic material aredegraded during the vegetation period, or NOM poor inthese specific structures are produced during the vegetationperiod. In all likelihood, both mechanisms apply. Therefore,the adverse potential against aquatic organisms (oxidativestress with consequent potential oxidation of membranes,fatty acids, amino acids, etc.) is considered to be highest inspring. In combination with hydrological events (quantitativeaspect of NOM input), the different qualities of NOM enter-ing lakes have a clear impact on the heterotrophic processesin non-eutrophicated lakes. For dystrophic Lake Örträsket(Sweden), Jansson et al.58 observed stimulated bacterial growthduring and after the spring flood. If our assumption ofthe highest adverse potential during spring times is valid, thepositive net effect of allochthonous NOM in Lake Örträsketmust be the result of adverse and beneficial effects, with signifi-cant dominance on the part of the gross heterotrophicpotential.

AbbreviationsFA, fulvic acid; HA, humic acid; HS, humic substances; NOM,natural organic matter isolated by RO; ‘NOM’, natural organicmatter isolated by ultrafiltration; OD, optical density; RO,reverse osmosis.

Fig. 6 Comparison of Φ∆ values from this work (grey symbols)with data from Haag et al.4 (open symbols) and Shao 56 (cited by Scullyet al.57; black symbols): (�) Lake Erie; (�) Sharpes Lake; (�) LakeSmithwick; (�) Fluka HA; (�) Black Lake HA; (�) Lake Baldegg; ( )Peat(S)-HA; ( ) Soil-FAII; ( ) Hiet-f.

Acknowledgements

Humic substances were kindly provided by E. Gjessing, R.Knauf, E. Zwirnmann, and T. Rossoll. Thanks to E. Zwirnmanfor the elemental analysis data, R. Ellerbrock for helpful discus-sions regarding the IR spectra, and Mrs G. Wöhlecke for excel-lent technical assistance.

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