Changes in Optical Properties Caused by UV-Irradiation of Aquatic

Feb 7, 2008 - Carlos, University of São Paulo, P.O. Box. 369,. 13560-970, São Carlos-SP, Brazil, Institute of Chemistry,. Paulista State University ...
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Environ. Sci. Technol. 2008, 42, 1948–1953

Changes in Optical Properties Caused by UV-Irradiation of Aquatic Humic Substances from the Amazon River Basin: Seasonal Variability Evaluation U R S U L A F A B I O L A R O D R Í G U E Z - Z Ú Ñ I G A , †,‡ DÉBORA MARCONDES BASTOS PEREIRA M I L O R I , * ,† W I L S O N T A D E U L O P E S D A SILVA,† LADISLAU MARTIN-NETO,† LUCIANA CAMARGO OLIVEIRA,§ AND JULIO CESAR ROCHA§ Embrapa Agricultural Instrumentation, P. O. Box. 741, 13560-970, São Carlos-SP, Brazil, Institute of Chemistry of São Carlos, University of São Paulo, P.O. Box. 369, 13560-970, São Carlos-SP, Brazil, Institute of Chemistry, Paulista State University “Julio Mesquita Filho”, P. O. Box. 355, 117800-900, Araraquara-SP, Brazil

Received August 28, 2007. Revised manuscript received December 9, 2007. Accepted December 20, 2007.

Aquatic humic substances (AHS) isolated from two characteristic seasons of the Negro river, winter and summer corresponding to floody and dry periods, were structurally characterized by 13C nuclear magnetic ressonance. Subsequently, AHS aqueous solutions were irradiated with a polychromatic lamp (290–475 nm) and monitored by its total organic carbon (TOC) content, ultraviolet–visible (UV–vis) absorbance, fluorescence, and Fourier transformed infrared spectroscopy (FTIR). As a result, a photobleaching up to 80% after irradiation of 48 h was observed. Conformational rearrangements and formation of low molecular complexity structures were formed during the irradiation, as deduced from the pH decrement and the fluorescence shifting to lower wavelengths. Additionally a significant mineralization with the formation of CO2, CO, and inorganic carbon compounds was registered, as assumed by TOC losses of up to 70%. The differences in photodegradation between samples expressed by photobleaching efficiency were enhanced in the summer sample and related to its elevated aromatic content. Aromatic structures are assumed to have high autosensitization capacity effects mediated by the free radical generation from quinone and phenolic moieties.

Introduction Dissolved organic matter (DOM) is the main constituent of the organic carbon pool in natural waters (1). It is also referred to as aquatic humic substances (AHS), in recognition of a similarity between freshwater DOM and soil organic matter (2). AHS are defined as heterogeneous mixtures of a variety of organic compounds, consisting of aromatic, aliphatic, phenolic, and quinolic functional groups with varying molecular sizes and properties (3). * Corresponding author phone: 55-16-3374-2477; fax: 55-16-33725958; e-mail: [email protected]. † Embrapa Agricultural Instrumentation. ‡ University of São Paulo. § Paulista State University “Julio Mesquita Filho”. 1948

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AHS contain groups (chromophores) that absorb sunlight and display the brown color often associated with water from wetlands (2). Absorption by these chromophores leads to structural modifications and gradual decomposition, called photodegradation with direct impact on the AHS ecological functions such as the control of surface water transparency and the nutrients availability for the aquatic microcosms (4). Direct photodegradation of AHS to carbon dioxide has been shown to play a significant role in the carbon budget (5, 6). Changes in the optical properties of AHS are quantitative manifestations of structural alterations, expressed by the photobleaching in the visible-UV (UV–vis) region. Thus, AHS spectroscopic properties and photodegradation parameters are relevant to better understand the DOM dynamic and its impact in riverine ecosystems. Special attention is focused on the Amazon basin because it is the greatest worldwide contributor of organic carbon in the ocean, with fluxes of DOM around 2.0 × 1013 g of C year-1 (6). In its huge system of tributaries, the Negro river represents the largest AHS input (4–10 g L-1 of AHS), reaching 70% of the DOM mainstream at its confluence with the Solimões river to form the Amazon (7). As defined by Latrubesse and Franzinelli (8), the Negro river is a “typical black” water river with olive to coffeebrown -colored water and transparencies from 1.3 to 2.3 m because of AHS content. It presents typical annual flooding regimes with an oscillation level of about 15 m. The highest discharges correspond to the months of June and July (winter season) and the lowest flow period to January and February (summer season) (9). The aim of this study is to investigate how the optical properties of the AHS are affected by UV irradiation. Additional evaluation was conducted considering the seasonal variability of their structure and the influence exerted on the photodegradation rates.

Materials and Methods Water Sampling and AHS Isolation. The sampling area was in the Negro river stream (Amazonas, Brazil) near the confluence of the Taruma Mirim and Taruma Açu rivers, GPS 3° 02′ 22′′S 60° 08′ 21′′W (Figure 1). Representative water samples for each month of 2003 were regularly collected in polyethylene bottles to a depth of 25 cm below the surface. They were immediately acidified (pH ) 3–4) and maintained in the dark at 4 °C until AHS extraction using the percolation methodology through the resin XAD 8, found in the Supporting Information (9). After extraction, the AHS samples were classified into groups in agreement with the typical climatic characteristics of the investigated region (10). Contrasting samples were chosen, with winter samples corresponding to the flooding season registered (June-July), while summer samples were taken during the river’s lowest level (December and January). 13C Nuclear Magnetic Resonance Measurements (13C NMR). Structural characterization and carbon functional group content were measured using solid-state 13C NMR using cross-polarization and magic angle spinning (CP/MAS) techniques. Spectra were recorded on a 400 MHz NMR Unity Inova Varian spectrometer. The aliphaticity and aromaticity degrees were calculated as explained in the Stevenson methodology (11); details are provided in the Supporting Information. Irradiation Experiments. Previous to the irradiation experiments, fluorescence tests at different AHS concentrations (0–100 mg L-1) were performed with two objectives: preventing inner filter effects and enabling acquiring a 10.1021/es702156n CCC: $40.75

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FIGURE 2. CP/MAS 13C NMR spectra of original winter and summer AHS without irradiation exposure. FIGURE 1. Map of the Negro river formation showing the sampling site used for this study. satisfactory spectroscopic signal. As a result, photodegradation rates were checked at 10 mg L-1, while structural evaluations were verified at 30 mg L-1. AHS solutions were prepared in MilliQ water and irradiated with a Hamamatsu lamp Lightningcure 200 (Hg-Xe) (290–475 nm). The medium light intensity over the samples was 5.8 mW cm-2, equivalent to four times the solar light on a sunny day in the Amazon region. For the first photodegradation tests, quartz flasks (250 mL) with AHS solution were irradiated for 2880 min with constant sampling of aliquots and stored until the UV–vis absorbance measurements. For the second set of tests, AHS solutions (40 mL) were subjected to different irradiation exposure times (0, 60, 120, 180, 300, 660, 1500, 2100, 3000, and 5000 min). Samples were evaluated for their total carbon concentration, fluorescence, and Fourier transformed infrared spectroscopies. All the irradiation experiments were performed in the dark, under aerobic conditions, continuous agitation, and temperature and pH control Optical Measurements. Absorbance scans (800–200 nm) were measured in a 1 cm quartz cell against an ultrapure MilliQ water blank using a 1601 PC UV–vis Shimadzu spectrophotometer. Observation wavelengths to monitor the photobleaching as a function of time were λ ) 254, 360, and 460 nm. Fluorescence spectra were generated with a Perkin-Elmer model LS50B equipped with a 150 W xenon arc lamp. The emission interval ranged between 300 and 600 nm and the excitation wavelength was 250 nm. Total Organic Carbon Content. The total organic carbon (TOC) content in the samples was determined by means of high-temperature catalytic combustion in a Shimadzu TOC5000 analyzer. Fourier Transformed Infrared Spectroscopy. The irradiated AHS solutions were freeze-dried and admixed with KBr to form the measurement discs. FTIR spectra were obtained in a Perkin-Elmer Spectrum 1000 spectrometer from 254 scannings and resolution of 4 cm-1 with baseline corrections at 4000, 2000, and 200 cm-1.

TABLE 1. Aromatic and Aliphatic Degrees of the Summer and Winter AHS Sampled in 2003 Obtained by 13C NMR

aromatics aliphatics

winter AHS, %

summer AHS, %

13.70 86.30

23.72 76.28

In that sense, summer AHS have an aromatic character 57% greater than winter samples. The first one corresponds to the dry season registering the lowest water level; therefore, there is no important incorporation of fresh organic matter to the river stream. In contrast, winter samples were collected during the rainy season when there is a constant percolation and incorporation of fresh organic matter, leading to the high aliphatic character. Irradiation Experiments. UV–Vis Absorbance Loss. As a consequence of irradiation, the absorbance spectra of AHS showed a significant absorbance loss in the near-UV and visible region (200–550 nm), with a noticeable change of the spectra format around 350 nm (Figure 3). The quantification of photobleaching was calculated from the following equation. % absorbance loss ) [(T Abs)t)0 (T Abs)t)2880] ⁄ ( T Abs)t)0 × 100 (1) where (T Abs)t)0 is the initial integrated spectra and (T Abs)t)2880 is the final integrated spectra (t ) 2880 min).

Results and Discussions Sample Characteristics. As observed in the CP/MAS 13C NMR spectra from the summer and winter nonirradiated AHS (Figure 2), they presented similar chemical structure; however, significative differences were found in their aromatic and aliphatic relative content (Table 1).

FIGURE 3. Irradiation effect after 48 h exposure in the absorbance UV–vis spectra. VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Normalized Percentages of Absorbance and TOC Losses due to the AHS Solutions Irradiation Exposure AHS sample

initial area

final area

absorbance loss (%)

initial TOC

final TOC

total mineralization (%)

winter summer

31.69 30.75

6.64 4.19

79.05 86.38

11.2 ( 0.2 10.9 ( 0.5

3.6 ( 0.1 2.3 ( 0.1

67.86 78.89

Table 2 registered important differences between samples, in which summer AHS registered the highest photobleaching. These differences may be associated with the samples’ photosensibilization in terms of their overall potential to generate free radicals from AHS (2, 12–15). Thus, the photodegradation of summer AHS, which has an elevated aromaticity, should be greater due to greater free radical generation from aromatic and oleofinic moieties. The rapid initial decrease of absorbance vs time follows a first-order exponential decay. Its linear fitting from eq 2 allows obtaining the degradation rates, enabling the predictio of the half-life times (13): ln[B ⁄ (At - A1)] ) kt

(2)

where k is the degradation rate (curve slope), A1 is the residual absorbance after irradiation, At is the absorbance at a given t time, and B is the difference between the initial and the residual absorbance. As observed in Figure 4 and the k values from Table 3, winter AHS present a faster rate. This behavior suggests that the aliphaticity character promotes a higher preliminary velocity derived from its inherent chemical instability, when compared to slower rates for summer AHS with a higher content of aromatic moieties. Notwithstanding, the results suggest that the faster process for the winter AHS would rapidly reach a maximum photobleaching limit, evidencing the formation of structures resistant to UV–vis effects. Wavelength Dependence. To detect the most affected portion of the absorption spectra, absorbance losses as a function of the irradiation exposures were calculated (Figure 5a,b). There was a nonuniform loss of absorbance heightened at shorter wavelengths. Thus, two same-intensity bands around 270 and 215 nm appeared at the beginning of the process. For longer times, at approximately 300 min, there was a progressive intensification of the 215 nm. The fact that the absorbance loss band shift does not correspond to the higher intensity lines of the lamp may be associated with the formation of excited states of AHS and reactive transients that participate in photochemical pathways for the destruction of components with broad absorption properties (5, 14). Although its identification was not the aim of this work, Del Vecchio and Blough (15) suggested phenomena affiliations to the presence of donor groups as polyhydroxylated aromatics derived from lignin, as well as phenols and indoles and acceptor groups such as quinones or other oxidized aromatics in the whole AHS structure. Moieties can produce donor–acceptor charge-transfer complexes, with broad absorption bands. These bands arise from the partial or complete promotion of an electron from donor to acceptor on the absorption of a photon (14, 15). Hence, photodegradation reactions might be involving two simultaneous pathways: the primary cleavage of compounds with energy bonds that match the irradiation wavelengths and the photodegradation mediated by intermediate reactive compounds, with a predominant production derived from the aromatic moieties (quinone and phenolic functionalities), more significant in the summer sample (5, 12). TOC Content. UV-induced DOM mineralization was observed from TOC diminution (Figure 6a). Decrements 1950

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FIGURE 4. Linear representation of the first-order exponential degradation of AHS at different wavelengths: (a) 254, (b) 360, and (c) 460 nm.

TABLE 3. Values of k Parameter Denoting Photodegradation Rates at Different Wavelengths AHS sample winter summer

the

wavelength (nm)

k × 10-4 (min-1)

R2

254 360 460 254 360 460

11.0 ( 0.3 13.0 ( 0.3 13.0 ( 0.6 10.0 ( 0.4 10.0 ( 0.1 11.0 ( 0.4

0.98 0.99 0.94 0.96 0.98 0.96

around 70% in the irradiated samples indicate the transformation of the organic fraction into inorganic carbon (CO2 and CO) and H2O (1, 16). The rapid initial decrement in TOC values and the significant and concurrent acidification (Figure 6b) might be influenced by the decomposition of the complex structure into low molecular weight organic compounds (1, 17–19) such as carbonyl and carboxylic acids, known as an important part of the AHS strong acidic functions (16–19) and the redissolution of the mineralized gases into the solutions in the form of carbonic acids (1, 19, 20). In addition, the lower losses of TOC values, when compared to the decreases in absorbance and fluorescence, suggest that the photodegradation results from the formation of carbon compounds that no longer absorb light to the same extent as the parent material (19). FTIR. Parts a and b of Figure 7 illustrate the FTIR spectra for the different irradiation times. The principal band changes were registered at 3100–3400 and 1560-1730 cm-1. The variable width between 3400 and 3100 cm-1, attributed to the O-H stretching (21), is related to polarization of the H in the hydroxyl radical. Since the AHS contain diverse

FIGURE 6. (a) TOC content and (b) pH variations as a function of irradiation time exposure of summer and winter AHS.

FIGURE 5. Time dependence of the absorption loss for AHS upon UV–vis irradiation exposure: (a) winter AHS and (b) summer AHS.

structural units joined together irregularly by covalent linkages of varying strengths, conformation and aggregational equilibrium are strongly influenced by hydrogen bonding (5). Decreasing intensities at 3400 cm-1 infer a loss of the weak-bonding forces (intermolecular H, dipole–dipole and dipole-ion bonds) originating a desegregation and intensification of the intramolecular bonds signal (21). Progressive changes in intensity and significant reduction of the band around 2950 cm-1 are associated with asymmetrical or symmetrical stretching of aliphatic bonds supporting the initial higher susceptibility of aliphatic structures. Moreover, the band at 1720–1740, attributed to the CdO stretching of carbonyl structures, decreases in intensity and also indicates the conversion of organic acids into carboxylate and CO2, after the ionization, and consequent stabilization of the pH (12, 22, 23). The following resolution improvement in the 1670 and 1632 cm-1 bands suggests the formation of less complex structures derived from the initial humic aggregate (1, 23, 24). Moreover, higher intensities between 1280 and 1200 cm-1 and 1000–1100 cm-1 corresponding to the ester and ether symmetric and asymmetric stretchings are also linked with the formation of photodegradation intermediates and its final mineralization, reported in the literature (22, 25). Fluorescence Spectroscopy. Parts a and b of Figure 8 show the emission spectra (λexc ) 250 nm) at different irradiation times. The initial spectra presented its maximum in the blue region (λ ) 430 nm), as a result of their high degree of structural conjugation and multifunctional properties (25, 26). VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 7. Comparison of AHS FTIR spectra corresponding to different irradiation times: (a) winter AHS and (b) summer AHS.

complexity produced by macromolecule fragmentation and rearrangement (1, 25). Additionally, the spectral areas of Figure 8 were calculated to monitor fluorescence during irradiation, showing the following two stages: An initial fast increase of the total fluorescence (0–660 min) quantified by 115 and 98% for winter and summer AHS, respectively. This stage corresponds to the fluorophore site activation and/or the possible formation of relatively simpler structures with greater fluorescent capacity (7, 26). A total fluorescence extinction (660–5000 min) with decrements of around 67 and 81% with relation to the winter and summer AHS initial spectra. These results are associated with the continuous degradation of fluorescent structures through the dissociation of conjugated and double bonds and diminution of the π electronic density (24–26). Finally, the highest fluorescence degradation for the summer AHS reinforces the most effective loss in UVabsorption derived from the higher degree of aromaticity, promoting a plentiful production of free radicals, derivatives from AHS photoionization and photooxidation. With regard to this, literature reports the main ones as being singlet oxygen, hydroxyl radical, and others (5, 12, 26). Generally, the results showed that both characteristic seasons of the Amazon region have a direct impact on the origin and thereby on the AHS structure. Winter AHS, representative of the floody season, demonstrated an elevated aliphatic character caused by continued incorporation of fresh organic matter from the superficial soil layers. Contrastingly, samples from the dry season (summer AHS) exhibited predominant aromatic elements derived from longer residence times in deeper soil layers. Likewise, the photodegradation behavior was seasonally dependent. The reduced photobleaching but faster rates of the winter AHS was a consequence of the most abundant aliphatic content. On the other hand, the summer AHS showed a greater degree of photodegradation mediated by slower rates and associated with a greater concentration of aromatic structures derived from its predominantly terrestrial origin and more stable nature. Bringing all the evidence together, the occurrence of two simultaneous processes can be inferred: a primary photolysis, involving the direct disruption of the molecule bonds whose energy corresponds to irradiation energy and is related to processes such as deprotonation and descarboxilation, evidenced by a decrease in the pH values and the rapid mineralization; a photolysis promoted by free radical species from quinone moieties, responsible for the most effective transformation and disruption of organic molecules. Thus, aromatic and more condensed structures with higher dissociation energies could be disaggregated and finally mineralized.

Acknowledgments FIGURE 8. Emission fluorescence spectra with excitation λ ) 250 nm at different irradiation times: (a) winter AHS and (b) summer AHS. The preliminary intensification of the signal up to approximately 660 min was explained by Patel-Sorrentino et al. (7) by the breaking of H bondings. The H+ release gives rise to a more spread out conformation, promoting activation of the initially hidden fluorophores in the aggregates. The bands’ progressive shifting to shorter wavelengths and the occurrence of a new band shifting in the relative violet region for the final spectra are consistent with new fluorescent and derived structures of lower molecular 1952

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We thank FAPESP (Project:99/09133-4), CEPOF (Project:98/ 14270-8), Brazilian Agencies, for financial support.

Supporting Information Available Further details on analytical methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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