Published: February 25, 2011

r 2011 American Chemical Society 3452 dx.doi.org/10.1021/es104288y | Environ. Sci. Technol. 2011, 45, 3452–3458

ARTICLE

pubs.acs.org/est

Effect of Humic Substance Photoalteration on Lead Bioavailabilityto Freshwater MicroalgaeJulian Spierings,‡ Isabelle A. M. Worms,‡ Pascal Mi�eville,§ and Vera I. Slaveykova*,†,‡

†Aquatic Biogeochemistry and Ecotoxicology, Institute F.-A. Forel, Faculty of Sciences, University of Geneva, 10, route de Suisse,1290 Versoix, Geneva, Switzerland‡Environmental Biophysical Chemistry, IIE-ENAC, Swiss Federal Institute of Technology (EPFL), Station 2, CH-1015 Lausanne,Switzerland§Laboratory of Biomolecular Magnetic Resonance, ISIC-SB, Swiss Federal Institute of Technology (EPFL), Station 6, CH-1015Lausanne, Switzerland

bS Supporting Information

’ INTRODUCTION

Bioavailability is a key concept linking the changes in toxicmetal concentration and speciation to their detrimental effectson biota.1 Among various freshwater constituents, humic sub-stances (HS) play an important role in buffering toxic metalconcentrations in aquatic ecosystems,2,3 thus reducing theirpotential detrimental effects on biota. Therefore, any environ-mental changes (e.g., climate and sunlight variability) that couldaffect HS concentration, structure, or reactivity can be expectedto alter (e.g., to decrease) the HS metal-binding properties, andthus reduce their toxicity to aquatic (micro)-organisms. It is welldocumented that sunlight, and in particular UV-B, affects DOMquality and quantity4,5 via different processes, such as miner-alization, photodegradation,6-8 or loss of suspended particulateorganic carbon and the production of dissolved organic carbon(e.g., by photodissolution).9 Nonetheless, there is a paucity ofstudies addressing the effects of HS photoalteration on thebioavailability of toxic metals to aquatic biota.

The limited literature shows that photolysis of DOM mostoften results in the release of the boundmetal, thus increasing thelevels of free metal ion concentrations.10-15 Exposure to UV-Blight of organic-rich riverine HS,13 alpine lake water samples,10,11

or purified peat humic acid15 resulted in increased free copper-ion concentrations. Increase of free cadmium-ion concentrationsvia photolytic release from cadmium-contaminated fulvic acidwas found, while no increase was observed in cadmium-amendednatural lake water HS.14 An enhancement of free lead-ionconcentrations in estuarine samples after UV-B irradiation wasreported.12 By contrast, light irradiation increased Cu complexa-tion by wetland HS and thus decreased free copper-ionconcentrations.6 Because metal bioavailability and biological

Received: December 21, 2010Accepted: February 10, 2011Revised: February 7, 2011

ABSTRACT: The present study provides results on the influ-ence of humic substance (HS) photoalteration on lead avail-ability to the freshwater microalga Chlorella kesslerii. Theevolution of the free lead-ion concentrations measured by theion exchange technique [Pb]IET and intracellular lead contentswas explored in the presence of Suwannee River humic (SRHA)and fulvic (SRFA) acids, as well as Aldrich humic acid (AHA)exposed at increasing radiance doses under a solar simulator.Modifications of HS characteristics highly relevant to Pbcomplexation and accumulation of HS to algal surfaces, includ-ing Fourier transform infrared spectroscopy, were followed. Itwas demonstrated that simulated sunlight exposure of HSincreased [Pb]IET in the medium for SRFA and SRHA, buthad no effect for AHA. No clear relationship was observed between the changes in free lead-ion concentrations and intracellularcontent in alga for all studied HS, suggesting that HS photodegradation products also exhibit Pb complexation properties, and thatdirect interactions between HS and alga are affected. Indeed, photoalteration of humic substances reduced the adsorption of HS tothe algal surface; the effect was more pronounced for SRFA and AHA and less significant for SRHA. The bioavailability results wereconsistent with the characterization of the phototransformation of humic substances: Pb speciation changes followed themodification of the relative abundance of the carboxylic groups and their molecular environment, while the reduced HS adsorptionto the alga correlated with losses of the double bond abundance and aromaticity.

3453 dx.doi.org/10.1021/es104288y |Environ. Sci. Technol. 2011, 45, 3452–3458

Environmental Science & Technology ARTICLE

effects are most often correlated with the concentration ofnoncomplexed metal, it can be anticipated that HS photoaltera-tion could increase metal bioavailability via an increase of freemetal-ion concentrations. Nonetheless, even more scarce andcontroversial is the literature about the biological availability(and toxicity) of metals in the presence of photoaltered DOM.Cu, Zn, Co, and Pb, but not Ni and Cd, toxicity to the freshwatergreen alga Pseudokirchneriella subcapitata increased up to 78%after 10 days of UV-B irradiation of high- and low-organiccarbon-water samples.16 Pb, Cu, Ni, and Cd concentrationsrequired to reduce algal growth by 50% were significantlydecreased with both solar radiation and UV-B treatment doses;however, the changes in toxicity with UV-dosing were incon-sistent among the metals tested.17 Cu toxicity to the larvaPimephales promelas increased in the presence of photooxidatedDOM, following model predictions based on free copper-ionconcentrations.6 Because DOM is a key parameter in waterquality and cumulates the action of different environmentalfactors,4 the understanding of DOM’s role inmetal bioavailabilityunder changing conditions will be of prime importance inreducing the uncertainty in assessing the toxic metal effect onbiota, and for the development of a predictive water qualityframework for changing environment.

Under well-controlled experimental conditions, the presentstudy explores the relationship between lead availability tofreshwater microalgae and Pb speciation in the presence of theHS exposed to solar radiation of increasing intensity. The specificquestions to address were: (i) To what extent does the sunlight-induced transformation of HS affect Pb bioavailability? (ii) Dobioavailability alterations follow the changes in free lead-ionconcentration? (iii) Is HS adsorption to alga altered by solarradiation, and what are the consequences for Pb bioavailability?Pb was chosen because of its toxicity and importance as apollutant, and also because of the more complex interactions inthe ternary system: Pb, microalgae, and HS.18,19

’MATERIALS AND METHODS

Standard fulvic and humic acids isolated from Suwannee River(SRFA and SRHA, International Humic Substances Society, St.Paul, MN) and Aldrich humic acid (AHA, Sigma-Aldrich Inc., StLouis, MO) were used as model HS. 1.0 g L-1 stock solutions ofHS were prepared in Milli-Q water from freeze-dried powders;pH was adjusted to 9.0 with diluted NaOH. Solutions werestored at 4 �C in the dark for at least 24 h to ensure equilibration.pH was then readjusted to 6.0 with ultrapure HNO3 (Baker,instra) before further dilution. HS were chosen because of theirdifferent aromaticity, fluorescence fingerprints (Table S1), num-ber, and affinity of the major metals binding sites.20

Exposure of HS to Solar Radiation of Increasing Intensity.Samples containing 10 mg C L-1 SRFA, SRHA, and AHA withpH adjusted to 6.0, in triplicate, were irradiated by the full solarspectrum for 12 h at increasing intensities under solar simulator(Sun 2000, Abet Technologies) equipped with a 1000 W Xesource lamp, an atmospheric absorption filter to mimic solarradiation distribution on Earth, and a light intensity controller.During the simulated sunlight exposure experiments, the radia-tion intensity was varied from 300 to 1000Wm-2. This variationcorresponded to a radiance doses range from 7 � 106 to 2.1 �107 J m-2 full solar spectrum (250-700 nm) and from 3 � 105

to 1 � 106 J m-2 UV-component (250-380 nm) measured atthe surface of the irradiated samples. The radiance doses were

chosen to correspond to the average annual cloudless sky UV-dose in Europe ranging from 8� 105 to 2� 106 J m-2.21 Duringthe experiments, the sample temperature was controlled byplacing the reactors in a cooled water bath. The pH of thesolutions was measured prior to and after the irradiation, and nosignificant changes were noted (ΔpH < 0.5 units).Pb Bioavailability to Freshwater Microalga Chlorella

kesslerii. Pb bioavailability was characterized by measuringthe intracellular Pb content, {Pb}int, in freshwater microalgaChlorella kesslerii exposed to 10-6 M Pb as Pb(NO3)2 for 1 h inthe presence of the 10 mg C L-1, irradiated HS, and nonirra-diated controls. To mimic highly impacted waters, 10-6 M totalPb concentration was chosen within the range of reported EC50

(50% effect concentration, i.e., growth inhibition) or 50% ofthe maximal {Pb}int for C. kesslerii.

22 {Pb}int was operationallydiscriminated from the surface bound Pb by ethylenediaminete-traacetic acid (EDTA, ultra Fluka) extraction.22 More detailsabout the experimental procedures can be found in the Support-ing Information.Determination of Free Pb Ion Concentration by Ion

Exchange Technique. The ion exchange technique (IET), inits “thermodynamic mode”, was used to determine free Pbconcentrations, [Pb]IET, under the conditions of bioavailabilityexperiments. The detailed experimental setup and procedures aredescribed elsewhere.23 Briefly, the ion exchange column usedcontained 50 mg of Dowex resin in Na form. An equilibrationtime of 2 h was used. [Pb]IET was determined from theconditional partitioning coefficient, λ0, following eq 1:24

½Pb�IET ¼ Vel � ½Pb�elλ0 � mres

ð1Þ

where [Pb]el (mol L-1) is the concentration of eluted metal,determined by inductively coupled plasma-mass spectrometry(ICP-MS, Perkin-Elmer, Elan DRC II);mres (g) is the amount ofresin, and Vel (L) is the elution volume. The conditionalpartitioning coefficient, λ0, was determined by using standardsolutions containing a known free lead-ion concentration and thesame cationic composition of the sample.Accumulation of Photoaltered HS to C. kesslerii. The

quantity of humic substances adsorbed to the algae was deter-mined as the difference between the HS concentrations in themedium prior to and after contact with algae, as detailedpreviously,25,26 at a contact time of 1 h. The concentration ofHS in the medium was measured by a UV spectrophotometer(Perkin-Elmer) at 254 nm. The absorbance measured at 254 nmwas converted to HS concentrations by using a calibration graphestablished for each HS and irradiation condition. A control ofHS-losses on the filters and container walls was performed underthe accumulation study conditions in the absence of algae andwas taken into account when determining the amount ofadsorbed HS. The amount of HS bound to algae was presentedas mg C per cm2 algal surface.Characterization of HS Photoalteration. To better under-

stand the observed effects of HS photoalteration to Pb speciationand bioavailability, possible modification of HS characteristicswas investigated by using large array of instrumental tools,probing different characteristics of these complex compounds.27

Potential photoalteration of major functional groups in the HSwas followed by the Fourier transform infrared spectroscopy(FT-IR). FT-IR spectra of HS exposed to simulated sunlight ofincreasing intensity were compared to the spectra of

3454 dx.doi.org/10.1021/es104288y |Environ. Sci. Technol. 2011, 45, 3452–3458

Environmental Science & Technology ARTICLE

nonirradiated control. The FT-IR frequency position was used toevaluate the chemical nature (chemical function) of photomo-difications, and the changes in the IR-transmittance (in %)relative to the nonirradiated controls were used to evaluate thevariation in the relative abundance of those chemical functions.General HS characteristics, such as specific UV absorbance,SUVA (L m-1 mg-1), fluorescence fingerprints, molar massdistribution, and dissolved organic carbon content (DOC), werealso determined. Experimental details about each instrumentaltechnique are provided in the Supporting Information.Data Analysis and Modeling. Control experiments (usually

performed in triplicate) in the presence of the nonirradiated HSwere always run in parallel with the assays in the presence of thephotoaltered HS. Statistical differences within [Pb]IET or {Pb}intdetermined in the presence of HS irradiated at increasing radiancedoses and nonirradiated controls were evaluated using the Stu-dent-Neuman-Keuls test in the Sigma Stat Software (SigmaStat, Chicago, IL). Experimentally determined intracellular con-tents were compared to those predicted by using the Michaelis-Menten equation and IET-measured lead concentrations,18,22 asdetailed in the Supporting Information.

’RESULTS AND DISCUSSION

Pb Complexation in the Presence of Photoaltered HS. Pre-exposure of HS to simulated sunlight of increasing intensityresulted in a significant increase in the IET-measured Pb con-centrations (Student-Neuman-Keuls test, P < 0.05) as com-pared to nonirradiated controls for both SRFA and SRHA.Enhanced [Pb]IET/[Pb]IET,CTR ratios of 2.2 and 2.4 were foundin the presence of SRFA or SRHA exposed to a radiant dose of2.8� 106 J m-2. For AHA, [Pb]IET was not affected significantly(Student-Neuman-Keuls test, P > 0.05) as compared to thenonirradiated controls (Figure 1). No clear relationship betweenthe radiance dose and the amount of the [Pb]IET was found for allHS. The raise in [Pb]IET was highest at 2.8� 106 Jm-2 exposure.Further increases in the radiance dose led to a decline in [Pb]IETreleased from the photoaltered HS; however, no statisticallysignificant difference (Student-Neuman-Keuls test, P > 0.05)was found for [Pb]IET in the presence of SRFA and SRHAexposed to doses of 8.6 � 106 or 2.1 � 107 J m-2. The lack of

correlation between the increase in [Pb]IET and radiance dosesuggests that photodegradation of humic substances in theaquatic environment can lead to the formation of degradationproducts with lead binding properties.Indeed, the evolution of the FT-IR spectra of the sunlight

exposed HS revealed an increase in the relative abundance of thecarboxyl groups in the photoaltered HS (see Characterization ofHS Photoalteration section) as compared to nonirradiatedcontrols. This is in agreement with literature reporting a forma-tion of pyruvate, glyoxylate, acetate, acetaldehyde, and formategroups in photooxidized DOM,28 all exhibiting metal-bindingproperties. Furthermore, these observations are also consistentwith a large chemical heterogeneity of the metal-binding sitestypical for HS, which can be altered to a different extent underirradiation.17 Overall, exposure of SRFA and SRHA to highsunlight radiance results in a decrease of their Pb bindingcapacity, as demonstrated by the bigger [Pb]IET; however, noclear relationship between the amount of the uncomplexed Pband the irradiation doses could be determined. Because thechanges of the intracellular Pb content are predicted to followfree lead-ion concentration variations in the exposure medium(e.g., biotic ligand model, BLM,29 a similar pattern of the {Pb}intvariations in the presence of the photoaltered HS was expected:for example, increase in {Pb}int in the presence of photoalteredSRFA and SRHA and no changes in the presence of AHA.Pb Availability to Microalgae in the Presence of Photo-

altered HS. Experimental results from bioassays demonstrated,however, that no correlation existed between the increasedrelease of Pb from the photoaltered HS (Figure 1) and intracel-lular Pb content in microalgae C. kesslerii (Figure 2). Pre-exposure of SRFA to sunlight of increasing intensity resulted ina decrease in the intracellular Pb content with respect to thenonirradiated control, opposite to about a 2-fold increase in thefree metal ion concentrations (Figure 2). A decrease in the{Pb}int was also observed in the presence of photoaltered AHA,although no significant changes in [Pb]IET were found. More-over, {Pb}int decreased in the presence of SRFA and AHAirradiated at increasing radiance doses, as illustrated by thereduction of {Pb}int/{Pb}int,CTR. By contrast, about 2.5-3-foldincrease of {Pb}int/{Pb}int,CTR was determined in the presenceof photoaltered SRHA, which followed (at least) qualitatively the

Figure 1. (A) IET measured lead concentrations (nonirradiated controls, [Pb]IET,CTR) in solution containing 10 mg C L-1 AHA, SRFA, or SRHAenriched with 1� 10-6 M Pb, pH = 6.0. (B) Effect of the radiant dose on [Pb]IET. The values were normalized to [Pb]IET,CTR. Error bars correspond tothe square root of the sum of the squares of the standard deviation for [Pb]ITE and [Pb]ITE,CTR, obtained from three replicates for each condition.Student-Neuman-Keuls test demonstrated significant differences between [Pb]IET measured in the presence of photoaltered SRFA and SRHA, whileno difference was found for AHA. IET-measured lead concentrations decreased in the order: [Pb]IET, SRFA > [Pb]IET, AHAg [Pb]IET, SRHA, prior to and[Pb]IET, SRFA g [Pb]IET, SRHA > [Pb]IET, AHA after irradiation.

3455 dx.doi.org/10.1021/es104288y |Environ. Sci. Technol. 2011, 45, 3452–3458

Environmental Science & Technology ARTICLE

increase in [Pb]IET. Furthermore, the intracellular Pb contentwas 9, 8, and 15 times superior to that predicted by the measured[Pb]IET (Figure 1) and models such as the BLM,29 for example,for AHA, SRFA, and SRHA, similarly to the nonirradiatedcontrols (Figure S1A). Because Pb uptake by C. kesslerii iscontrolled by the transport across the biological membranerather than by the diffusion in the medium (e.g., diffusive fluxof free lead-ions much greater than the internalization flux18,22),the discrepancy betweenmeasured and predicted intracellular Pbcontents cannot be assigned to the contribution (e.g., viadissociation) of labile Pb-organic matter complexes formedafter HS photoalteration. Furthermore, the increasing radiancedoses reduced the shift between the measured and predicted Pbintracellular contents for AHA and SRFA, but had no significanteffect for SRHA (Figure S3B).These observations are in agreement with previous studies

about the bioavailability of Pb to microalgae in the presence ofdissolved18,25,26,30 and colloidal organic matter of differentcomposition,23 demonstrating that HS could also affect (e.g.,increase) Pb bioavailability by adding supplementary bindingsites26,30 and affecting the algal cell wall speciation.18 Theseeffects relate to the capacity of HS to accumulate on microorgan-ism surfaces.31 Accordingly, the hypothesis that the photoaltera-tion of HS influences (e.g., reduce) the capacity of HS toaccumulate to algal surfaces was further tested experimentally.Accumulation of Photoaltered HS to Algal Surface. Re-

duced amounts of the HS accumulated to C. kesslerii afterirradiation at 2.1 � 107 J m-2 were measured as compared tothe nonirradiated controls (Figure 3). Prior to irradiation, theamount of adsorbed HS to C. kesslerii was comparable for SRFAand AHA and higher for SRHA: {HA}ads/[HS]diss ratios were8.9 ( 1.0, 8.7 ( 1.0, and 11.2 ( 0.5 (�10-3 cm). A radiancedose of 2.1 � 107 J m-2 resulted in a reduction of the ratio{HA}ads/[HS]diss to 2.0 ( 0.4, 1.45 ( 0.2, and 8.0 ( 0.5(�10-3 cm) for AHA, SRFA, and SRHA, respectively, clearlyindicating that photoalteration resulted in a decrease of theamount of HS adsorbed to algae in a HS-specific way.The above results demonstrate that the consequences of

sunlight irradiation of organic matter on Pb availability by themicroalga C. kesslerii are complex and that, in addition to thecomplexation properties of HS, solar radiation also alters theircapacity to interact (e.g., to adsorb) with algae. To our knowl-edge, this is a first study demonstrating the effect of the solar

radiation on HS0 capacity to adsorb to microalgae. An increase inthe [Pb]IET is expected to enhance {Pb}int, while the reduction ofthe amount of HS adsorbed to C. kesslerii results in a decrease of{Pb}int for a given free lead-ion concentration. To link the aboveobservations and elucidate the causal relationship with photo-induced HS structure modifications, the influence of the irradia-tion of HS on different functional groups was further evaluated.In particular, the focus was on the alteration of carboxylic groupsand their environment, as well as the aromatic structuresconsidered of high relevance to Pb complexation and HSadsorption to algae, respectively. Carboxylic groups are consid-ered to be the major metal complexing groups in HS undercircumneutral pH. Their chemical behavior is highly dependenton their structural environment such as the type and position ofother chemical groups: for example, ketones and hydroxyl groupsthat may participate with carboxylic groups in metal chelation.32

The adsorption of HS to algae is dependent on the HScomposition and seems to be significant for HS with a highhydrophobic character (and high aromaticity).31 Therefore,exploring the effect of sunlight on the aromatic structures wasalso of interest.

Figure 2. (A) Intracellular lead content in the presence of 10 mg C L-1 AHA, SRFA, or SRHA (nonirradiated controls, {Pb}int,CTR). Total leadconcentration is 10-6 M, pH = 6.0. (B) Effect of the radiance dose on {Pb}int in the presence of 10 mg C L-1 AHA, SRFA, and SRHA irradiated atincreasing doses. {Pb}int were normalized to {Pb}int,CTR. Error bars correspond to the square root of the sum of the squares of the standard deviation for{Pb}int and {Pb}int,CTR, obtained from three replicates for each condition. In the absence of HS, {Pb}int = (3.6( 0.4)� 10-12 mol cm-2 for C. kessleriiexposed to 1 � 10-6 M Pb for 1 h, pH = 6.0.

Figure 3. Adsorbed HS onto algal cells prior to and after irradiation at2.1 � 107 J m-2 full solar spectrum, pH 6.0. Error bars correspond tostandard deviations of three replicates.

3456 dx.doi.org/10.1021/es104288y |Environ. Sci. Technol. 2011, 45, 3452–3458

Environmental Science & Technology ARTICLE

Effect of Solar Radiation on Relevant HS Characteristics.FT-IR revealed differences between the nonirradiated samplesand those receiving increasing radiation doses (Figure 4). Tworegions of interest with respect to photoalteration of Pb-com-plexing properties and the adsorption of the HS to the algae wereconsidered: 3500-2500 and 1700-1000 cm-1. The first spec-tral window is abundant in H-bonded OH stretching, aromatic,and aliphatic C-H stretching.33 The second spectral window isrich in CdO stretch from COOH and asymmetrical andsymmetrical (1650-1590 cm-1) stretching from deprotonatedcarboxylic groups (1650-1590 cm-1) and amides.33,34 A com-mon and prominent feature of all three HS was that the relativeabundance of the deprotonated carboxylic and amide groupsincreased upon irradiation, as illustrated by the band at 1622-1597 cm-1 attributed to the CdO stretch from COO- andamide groups. Given that nitrogen represents only 1.7% in SRHAand only 0.72% in SRFA,20 the intensity of transmittance changesseems inconsistent with amide group assignment. Thus, wesuppose that CdO stretch originates from COO- rather thanfrom amide groups. Furthermore, it was found that the amidegroups are very prone to photooxidation.5

The formation of hydroxyl groups from carboxylic acids andalcohols was also observed from the evolution of the broadbands, which appeared close to 3400 cm-1 for SRFA and3309 cm-1 for SRHA, usually associated with the H-bondedOH stretching. This was most evident for SRHA (25% increasein the transmittance) rather than for SRFA (only 3-7% in-crease). In addition, the photoalterations were not directlyproportional to the radiance dose, but rather appeared at lowerdoses. For example, the aldehyde groups formed at lowerradiance doses and decreased when radiance doses furtherincreased (e.g., photooxidation of aldehydes in acids). Thepercentage of newly formed carboxyl groups (probably fromlow molar mass acids) increased with the increased radiancedoses for SRFA, did not change significantly for SRHA, butdecreased for AHA. An opposite tendency was observed in theevolution of the newly formed hydroxyl groups: enhancedradiance doses resulted in a decrease of the % of OH-groupsfor SRFA, no changes for SRHA, and an increase for AHA. At thehighest radiance doses, a formation of free (non H-bonded) OHwas observed for AHA. A formation of both aldehyde and arenefunctional groups was found only in photoaltered SRHA ascompared to nonirradiated control.Another feature of the FT-IR spectra evolution with the

irradiation intensity, relevant with respect to the adsorption ofHS to algae, was the important decrease in the relative abundanceof the double bonded structures CdC, spectral band at2852 cm-1. These modifications are most visible in the FT-IRspectra of SRFA (Figure 4B) in which aromatic carbon repre-sents about 30% of the total organic carbon.36 FT-IR spectraevolution is also consistent with the SUVA and fluorescentfingerprint changes upon irradiation (Table S1 and Figure S2).UV-absorbance declines in the wavelength range from 250 to450 nm after irradiation, with HS-absorbance losses higher at325 nm than at 254 nm. Upon irradiation at 2.8 � 106 J m-2,a 10-15% increase in A254/A325nm was found, while theSUVA-values increased significantly in an HS-specific manner.At the highest radiance doses used in the present study (2.1 �107 J m-2), the SUVA-rise was ca. 40% for SRFA, 17% for SRHA,and 14% for AHA, as compared to the nonirradiated controls.Decreased absorbance in the UV region suggest a loss ofsp2-hybridized carbon bonds (e.g., double bonds in aromatics),

which is consistent with the significant decrease in the percentageof the double bonds in FT-IR for SRFA. However, FT-IR spectrado not show significant variations in sp2-double bonds for SRHA,although the important decrease in SUVA (and aromaticity).SUVA determined at 254 nm is highly correlated to thearomaticity as determined by the 13C NMR different types ofsource materials;37 however, a wide range of reactivity for DOMwith similar SUVA was observed, showing that SUVA does notprovide information about the reactivity of DOM derived fromdifferent types of source materials.A decrease in the HS adsorption to algae upon phototrans-

formation of HS is in qualitative agreement with a decrease in thearomaticity, as shown by SUVA evaluation (Table S1). Decreasein SUVA and UV absorbing properties in combination with nochanges in the DOC concentrations (Table S1) are highlysuggestive of aromatic moieties conversion to low molar massorganic compounds (Figure S3).For SRHA and SRFA, the ratio of the intensity of peaks “type

A-humic” and “type C-fulvic” (F240/445/F320/445) augmented

Figure 4. Fourrier transform infrared (FT-IR) spectra of AHA (A),SRFA (B), and SRHA (C). For all spectra, the relative percenttransmittance is plotted against wavenumber. Band frequency assign-ment based on peaks previously reported for the humic substances:33,35

3400-3200 cm-1, OH stretch from -COOH and -COH; 3100-2800 cm-1, CH stretch from CH, -CH2, and CH3 and aromatics;2700-2400 cm-1, OH stretch from strongly H-bonded -COOH;1622-1597 cm-1, CdO stretch from COO- and amide groups. Notethat the position of the CdO stretch from the carboxylic groups, usuallyobserved at 1700 cm-1, is pH dependent and shifts toward high energieswhen pH increases.34

3457 dx.doi.org/10.1021/es104288y |Environ. Sci. Technol. 2011, 45, 3452–3458

Environmental Science & Technology ARTICLE

with the increase of radiance dose, suggesting that fulvicfluorophores are more sensitive to irradiation than humicones (Figure S2 and Table S1). Similarly, the fraction F240/445/F320/445 increased after a 20 min exposure to UV-visibleradiation of Amazon affluent waters.38 Decreases in the aroma-ticity (e.g., decrease in the quinine and lignin derived poly-phenoles) with increasing irradiation doses also suggests that themolecular electronegativity, related to the electron-withdrawingcharacter of the aromatic moieties, will decrease and thus couldreduce the complexation of the positively charged Pb2þ.27

Indeed, the increase in Pb released from SRFA was morepronounced than that from photoaltered AHA.It was outside of the scope of the study to explore the detailed

compositional, structural, and functional characteristics of HSand propose the transformation pathways. Nonetheless, theresults of the present study are consistent with the extensiveliterature (see ref 5 for review) demonstrating that irradiatedHS acts as photosensitizers, generating short-lived, highlyreactive oxygen species. The above observations also confirmedthat AHA is less prone to photooxidation as compared to SRFAand SRHA; consequently, Pb speciation and uptake wereinsignificantly affected. The obtained results also suggest thatthe influence of sunlight radiation affects Pb complexation byboth alteration of the relative abundance of carboxylic groupsand by affecting their molecular environment and thus theirPb affinity in a HS-specific way. Therefore, in addition to thedirect deleterious influence of UV-B exposure on the aquaticphytoplankton,39 an increased sunlight radiation can signifi-cantly affect contaminant biouptake by changes in the mediumchemistry. DOM is a key water quality parameter encompassingthe influence of rarely monitored processes related to globalenvironmental changes. Thus, our ability to interpret andpredict the impact of toxic metals on aquatic phytoplankton,representing the important primary producers, under changingconditions is directly related to our ability to quantify andforesee the effect of dissolved organic matter on metalbioavailability.

’ASSOCIATED CONTENT

bS Supporting Information. Excitation-emission matrices,FT-IR results, and AFlFFF fractograms of HS irradiated withsunlight of increasing doses, as well as experimental details aboutHS characterization, modeling, and prediction of intracellular Pbcontent in Chlorella kesslerii. This material is available free ofcharge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Phone:þ41 22 379 03 35. Fax:þ41 22 379 03 29. E-mail: vera.slaveykova@unige.ch.

’ACKNOWLEDGMENT

We gratefully acknowledge the financial support providedby the Swiss National Science Foundation project PP002-102640. Thanks are extended to T. Kohn for kindly providingaccess to the solar simulator, E. Alasonati for the AFlFFF-UVmeasurements, and J. Bolzman for HS adsorption to algaeexperiments.

’REFERENCES

(1) Wilkinson, K. J.; Buffle, J. Critical evaluation of physicochemicalparameters and processes for modelling the biological uptake of tracemetals in environmental (aquatic) systems. In Physicochemical Kineticsand Transport at Biointerfaces; van Leeuwen, H. P., Koester, W., Eds.;John Wiley & Sons: Chichester, 2004; pp 447-533.

(2) Slaveykova, V. I.; Wilkinson, K. J. Predicting the bioavailability ofmetals and metal complexes: Critical review of the biotic ligand model.Environ. Chem. 2005, 2, 9–24.

(3) Campbell, P. G. C.; Errecalde, O.; Fortin, C.; Hiriart-Baer, W. R.;Vigneault, B. Metal bioavailability to phytoplankton - applicability of thebiotic ligand model. Comp. Biochem. Physiol., C 2002, 133, 189–206.

(4) Zepp, R. G.; Erickson, D. J., III; Paul, N. D.; Sulzberger, B.Interactive effects of solar UV and climate change on biogeochemicalcycles. Photochem. Photobiol. Sci. 2007, 6, 286–300.

(5) Sulzberger, B.; Durisch-Kaiser, E. Chemical characterization ofdissolved organic matter (DOM): A prerequisite for understanding UV-induced changes of DOM absorption properties and bioavailability.Aquat. Sci. 2009, 71, 104–126.

(6) Brooks, M. L.; Meyer, J. S.; McKnight, D. M. Photooxidation ofwetland and riverine dissolved organic matter: Altered copper com-plexation and organic composition. Hydrobiologia 2007, 579, 95–113.

(7) Moran, M. A.; Sheldon, W. M.; Zepp, R. G. Carbon loss andoptical property changes during long-term photochemical and biologicaldegradation of estuarine dissolved organic matter. Limnol. Oceanogr.2000, 45, 1254–1264.

(8) Obernosterer, I.; Benner, R. Competition between biologicaland photochemical processes in the mineralization of dissolved organiccarbon. Limnol. Oceanogr. 2004, 49, 117–124.

(9) Mayer, L. M.; Schick, L. L.; Skorko, K.; Boss, E. Photodissolutionof particulate organic matter from sediments. Limnol. Oceanogr. 2006,51, 1064–1071.

(10) Sander, S.; Kim, J. P.; Anderson, B.; Hinter, K. A. Effect of UVBirradiation on Cu2þ- binding organic ligands and Cu2þ speciation inalpine lake water of New Zealand. Environ. Chem. 2005, 2, 56–62.

(11) Shank, G. C.; Whitehead, R. F.; Smith, M. L.; Skrabal, S. A.;Kieber, R. J. Photodegradation of strong copper complexing ligands inorganic-rich estuarine waters. Limnol. Oceanogr. 2006, 51, 884–892.

(12) Lindim, C.; Mota, A. M.; Goncalves, M. L. S. Influence of UV-Bradiation in lead speciation from an estuarine sample. Water Res. 2000,34, 3325–3334.

(13) Brooks, M. L.; Meyer, J. S.; Boese, C. J. Toxicity of copper tolarval Pimephales promelas in the presence of photodegraded naturaldissolved organic matter. Can. J. Fish. Aquat. Sci. 2007, 64, 391–401.

(14) Neron, R.; Auclair, J. C.; Fortin, C. Rate of Cd2þ release fromdissolved fulvic acid and natural dissolved organic carbon as a function ofUVB dose. Environ. Chem. 2006, 3, 433–438.

(15) Vidali, R.; Remoundaki, E.; Tsezos, M. Humic acids copperbinding following their photochemical alteration by simulated solarlight. Aquat. Geochem. 2010, 16, 207–218.

(16) Winch, S.; Ridal, J.; Lean, D. Increased metal bioavailabilityfollowing alteration of freshwater dissolved organic carbon by ultravioletB radiation exposure. Environ. Toxicol. 2002, 17, 267–274.

(17) Winch, S.; Lean, D. Comparison of changes in metal toxicityfollowing exposure of water with high dissolved organic carbon content tosolar, UV-B andUV-A radiation.Photochem. Photobiol. 2005, 81, 1469–1480.

(18) Lamelas, C.; Pinheiro, J. P.; Slaveykova, V. I. Effect of humicacid on Cd(II), Cu(II), and Pb(II) uptake by freshwater algae: Kineticand cell wall speciation considerations. Environ. Sci. Technol. 2009,43, 730–735.

(19) Lamelas, C.; Slaveykova, V. I. Pb uptake by the freshwater algaChlorella kesslerii in the presence of dissolved organic matter of variablecomposition. Environ. Chem. 2008, 5, 366–372.

(20) Averett, R. C.; Leenheer, J. A.; McKnight, D. M.; Thorn, K. A.Humic substances in the Suwannee River, Georgia: interactions, proper-ties, and proposed structures. U.S. Geol. Surv. Water-Supply Pap.1994, 2373.

3458 dx.doi.org/10.1021/es104288y |Environ. Sci. Technol. 2011, 45, 3452–3458

Environmental Science & Technology ARTICLE

(21) Seckmeyer, G.; Darius, P.; Merle, G.; Diamantino, H.; Bjorn, J.;Ann, W.; Anna-Maria, S.; Alkis, B.; Berit, K.; Colette, B.; Jacqueline, L.;Brian, G.; Peter, K.; Tapani, K.; Jussi, K.; Beate, U.; Harry, S.; Peter den,O.; Michal, J.; Peter, W.; Julian, G.; Bernhard, M.; Alain de la, C.; Stana,S.; Fernanda, C. Variability of UV irradiance in Europe. Photochem.Photobiol. 2008, 84, 172–179.(22) Slaveykova, V. I.; Wilkinson, K. J. Physicochemical aspects of

lead bioaccumulation by Chlorella vulgaris. Environ. Sci. Technol. 2002,36, 969–975.(23) Worms, I. A.M.; Traber, J.; Kistler, D.; Sigg, L.; Slaveykova, V. I.

Uptake of Cd(II) and Pb(II) by microalgae in presence of colloidalorganic matter from wastewater treatment plant effluents. Environ.Pollut. 2010, 158, 369–374.(24) Cantwell, F. F.; Nielsen, J. S.; Hrudey, S. E. Free nickel ion

concentration in sewage by an ion exchange column-equilibrationmethod. Anal. Chem. 1982, 54, 1498–1503.(25) Slaveykova, V. I.;Wilkinson, K. J.; Ceresa, A.; Pretsch, E. Role of

fulvic acid on lead bioaccumulation by Chlorella kesslerii. Environ. Sci.Technol. 2003, 37, 1114–1121.(26) Lamelas, C.; Wilkinson, K. J.; Slaveykova, V. I. Influence of the

composition of natural organic matter on Pb bioavailability to micro-algae. Environ. Sci. Technol. 2005, 39, 6109–6116.(27) Leenheer, J. A.; Crou�e, J. P. Characterizing aquatic dissolved

organic matter. Environ. Sci. Technol. 2003, 37, 18A–26A.(28) Lepane, V.; Persson, T.; Wedborg, M. Effects of UV-B radiation

on molecular weight distribution and fluorescence from humic sub-stances in riverine and low salinity water. Estuarine, Coastal Shelf Sci.2003, 56, 161–173.(29) Di Toro, D. M.; Allen, H. E.; Bergman, H. L.; Meyer, J. S.;

Paquin, P. R.; Santore, R. C. Biotic ligand model of the acute toxicity ofmetals. 1. Technical basis. Environ. Toxicol. Chem. 2001, 20, 2383–2396.(30) Lamelas, C.; Slaveykova, V. I. Comparison of Cd(II), Cu(II),

and Pb(II) biouptake by green algae in the presence of humic acid.Environ. Sci. Technol. 2007, 41, 4172–4178.(31) Campbell, P. G. C.; Twiss, M. R.;Wilkinson, K. J. Accumulation

of natural organic matter on the surfaces of living cells: implications forthe interaction of toxic solutes with aquatic biota. Can. J. Fish. Aquat. Sci.1997, 54, 2543–2554.(32) Leenheer, J. A.; Wershaw, R. L.; Reddy, M. M. Strong-acid,

carboxyl-group structures in fulvic acid from the Suwannee River,Georgia. 2. Major structures. Environ. Sci. Technol. 1995, 29, 399–405.(33) Davis, W. M.; Erickson, C. L.; Johnston, C. T.; Delfino, J. J.;

Porter, J. E. Quantitative Fourier transform infrared spectroscopicinvestigation humic substance functional group composition. Chemo-sphere 1999, 38, 2913–2928.(34) Cabaniss, S. E.; Leenheer, J. A.; McVey, I. F. Aqueous infrared

carboxylate absorbances: aliphatic di-acids. Spectrochim. Acta, Part A1998, 54, 449–458.(35) Elkins, K. M.; Nelson, D. J. Fluorescence and FT-IR spectro-

scopic studies of Suwannee river fulvic acid complexation with alumi-num, terbium and calcium. J. Inorg. Biochem. 2001, 87, 81–96.(36) McKnight, D. M.; Boyer, E. W.; Westerhoff, P. K.; Doran, P. T.;

Kulbe, T.; Andersen, D. T. Spectrofluorometric characterization ofdissolved organic matter for indication of precursor organic materialand aromaticity. Limnol. Oceanogr. 2001, 46, 38–48.(37) Weishaar, J. L.; Aiken, G. R.; Bergamaschi, B. A.; Fram, M. S.;

Fujii, R.; Mopper, K. Evaluation of specific ultraviolet absorbance as anindicator of the chemical composition and reactivity of dissolved organiccarbon. Environ. Sci. Technol. 2003, 37, 4702–4708.(38) Mounier, S.; Patel, N.; Quilici, L.; Benaim, J. Y.; Benamou, C.

Three-dimensional fluorescence of the dissolved organic carbon in theAmazon river. Water Res. 1999, 33, 1523–1533.(39) Haeder, D. P.; Kumar, H. D.; Smith, R. C.; Worrest, R. C. Effect

of solar UV radiation on aquatic ecosystems and interactions withclimate change. Photochem. Photobiol. Sci. 2007, 6, 267–285.