Environ. Sci. Technol. 2006, 40, 3717-3722
A Multifactor Exploration of the Photobleaching of Suwannee River Dissolved Organic Matter Across the Freshwater/Saltwater Interface KELLY H. HEFNER, JUSTINA M. FISHER, AND JOHN L. FERRY* Department of Chemistry and Biochemistry, University of South Carolina Columbia, South Carolina 29208
A four factor central composite experimental design was applied to explore the photobleaching of Suwannee River dissolved organic matter (SRDOM) at 350 nm as a function of the tetravariate system of [SRDOM], total [Fe(III)], [NO3-], and salinity. The ranges of each factor were set to cover their likely concentrations at the freshwater/ saltwater interface, to encompass the possible conditions encountered during the transition from the terrestrial to marine environment. Each experiment was carried out using a minimum of 25 different initial conditions, with 3-6 replicates/condition. The resulting data set mapped out the effects of multiple photoactive components on the rate of photobleaching. Under the conditions tested (nominally total [Fe(III)] 0.00-4.00 µM; [NO3-] 0.00-60.00 µM; SRDOM 0.00-30.00 mg/L; salinity 0.00-35.00 ppt, polychromatic illumination, pH 8.2) all samples photobleached at all wavelengths measured, and the absorption at 350 nm bleached the most rapidly. The most important factor for predicting the rate of photobleaching at 350 nm was the initial loading of SRDOM; the effect of all other factors on photobleaching was not significant at the 95% level of confidence. Varied salinity, Fe(III), or added D2O had no effect on the rate of photobleaching, indicating that hydroxyl radical, singlet oxygen, and superoxide did not contribute significantly to the loss of the chromophore at 350 nm. The addition of hydroquinone or thiosulfate inhibited photobleaching, suggesting photobleaching may depend on a weaker oxidant such as the excited-state acceptors (derived from SRDOM directly) produced during photoinitiated charge-transfer processes. The advantages of multifactor experimental techniques for exploring SRDOM photochemistry are discussed.
Introduction Terrestrially derived dissolved organic matter (DOM) is primarily a mixture of degraded biomacromolecules with trace amounts of anthropogenic chemicals (1-5). The chromophoric components of DOM play an important role in environmental aquatic systems by moderating the photochemical degradation of trace organics, affecting light available for photosynthesis, generating transient reactive species, etc. (3, 6-13). The DOM absorption spectrum (over * Corresponding author phone: (803)777-2646; fax: (803)777-9521; e-mail:
[email protected]. 10.1021/es052513h CCC: $33.50 Published on Web 05/06/2006
2006 American Chemical Society
the range 290-800 nm) typically features a strong absorption at 290 nm that exponentially decays to near zero at approximately 500 nm. Some fraction of this absorbance is clearly a function of a linear combination of discrete absorbances from well-defined molecular chromophores. However, recent work suggests that more of the absorbance characteristics of DOM are the product of the interaction of a large manifold of optical charge-transfer bands (10, 14, 15). DOM is known to undergo photobleaching through direct photolysis (photoionization or fragmentation) and also through the reaction of chromophoric components with reactive oxygen species such as 1O2, HO•, alkylperoxyl radicals, H2O2, etc. (9-12, 14, 16-20). The latter can be generated directly from the photoexcitation of DOM and also from excitation of common surface water constituents such as NO3- or Fe(III) (1, 2, 10, 12, 14-28). However, their potential impact on photobleaching is difficult to estimate, as they also react with many inorganic ions such as HCO3-/CO32-, I-, and Br- (ions often associated with salinity). The implication is that intrinsic properties of DOM and extrinsic solution parameters (initial [DOM], [NO3-], [Fe(III)], and salinity) related to water quality all participate in determining the rate of photobleaching, making it an environmental process that is essentially dependent on local conditions. The focus of this research was to develop an approach for exploring SRDOM photobleaching that quantified the effects of these four factors, along with all possible interfactor effects, under conditions that encompassed the widest possible variety of routinely encountered surface waters. Suwannee River DOM (SRDOM) was used because it is a widely studied reference material with very similar photochemical properties to several different surface waters (10, 29-36). The specific goal was to test the hypothesis that the factors [SRDOM], [NO3-], [Fe(III)], and salinity influence photobleaching by a combination of additive and synergistic effects and ideally to assess the magnitude of that influence. The concentration ranges for each component were chosen based on their concentration levels reported in natural waters, erring high for the sake of wider applicability (2, 4, 37-41). The rate of SRDOM photobleaching was indexed against the four factors by applying a combinatorial photochemical approach based on the central composite experimental design. The central composite approach was chosen because it is particularly well-suited for detecting possible interfactor contributions with high resolution across factor levels while minimizing the total number of conditions required for robust statistical analysis (42, 43). The approach highlighted the role of SRDOM in its own photobleaching and suggests that this factor may be the most important determinant of photobleaching for other surface waters as well.
Experimental Methods Materials. All solutions were prepared with Barnsted E-Pure 18 MΩ water. SRDOM (IHSS 1R101N) was collected using ultrafiltration, and its characterization (provided by the IHSS) is given in Supporting Information Tables 1-4. SRDOM was contaminated with 8.83 µg of native Fe/mg (44). Sodium nitrate (99.1%), sodium bicarbonate (100%), and benzoic acid (99.5%) were used as received from Fisher Scientific. Fe2(SO4)3‚5H2O (97%), hydroquinone (98%), and sodium thiosulfate (99%) were purchased from Aldrich and used as received. Instant Ocean (Supporting Information Table 5; manufactured by Aquarium Systems, Inc.) synthetic sea salt was used to adjust salinity after purification by passing through C18 silica gel. Deuterium oxide (D, 99.9%) was purchased from Cambridge Isotope Laboratories, Inc. Zinsser VOL. 40, NO. 12, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3717
TABLE 1. Experimental Conditionsd factor concentration levelsa factor (units)
-2c
-1c
0c
1c
2c
factor x1: Fe(III) (µM)b factor x2: NO3- (µM) factor x3: SRDOM (mg/L) factor x4: salinity (ppt)
0 0 0 0
1.00 14.70 7.50 8.75
2.00 29.50 15.00 17.50
3.00 44.20 22.50 26.25
4.00 59.00 30.00 35.00
a Denotes initial concentrations. b Nominal concentration. c Coded factor levels. d Factors, variable codes, and concentrations for the fourfactor central composite design; n ) 3 for axial and factorial point experiments, n ) 6 for center point experiments.
Analytic solid glass 96 well microplates were acquired from Spike International Ltd. All solutions were stored in the dark. Experimental Design. A central composite design was used to explore the effects of the four factors (SRDOM, Fe (III)aq NO3-, salinity) addressed by this study. The design allowed the performance of parallel experiments for up to 25 different environmental simulations simultaneously. Five concentration levels of each factor were included. An experiment with 0.00 mg/L [SRDOM]o was included in the experimental design as a control. There were six replicates of the center point condition (2.00 µM Fe(III), 29.50 µM NO3-, 15.00 mg/L SRDOM, 17.50 ppt salinity) and triplicates of the axial and factorial point conditions. A total of 78 experiments with 25 different initial conditions (Table 1) were generated using Design Expert, Version 5.0.3, Stat-Ease, Inc. This software was also used in all data analysis. Seven sets of these 78 experiments were prepared to cover timed irradiation periods (t ) 0, 5, 10, 20, 30, 40, 50 h). Mechanism probe experiments were conducted in 50% D2O (2.00 µM Fe, 31.00 µM NO3-, 16.00 mg/L SRDOM, 15.00 g/L salinity), with 1.00 mM Na2S2O3 (2.00 µM Fe, 17.50 µM NO3-, 15.00 mg/L SRDOM, 32.00 g/L salinity), or 1.00 mM hydroquinone (2.00 µM Fe, 17.50 µM NO3-, 15.00 mg/L SRDOM, 32.00 g/L salinity). Actinometry. Chemical actinometry was performed with the nitrate/benzoic acid actinometer (45) to gauge the linearity of lamp output over the course of the experiments. Electronic actinometric measurements (Atlas Sunchex Sensor) were used to characterize the homogeneity of the light field (measuring the band from 295-400 nm) at positions throughout the sample area of the Suntest. A steel mesh diffuser was applied to homogenize the light field and reduce the intrinsic intensity variation to an RSD of 7.0% at a spatial resolution of 4.0 cm2. Overall lamp output (W/m2) was determined by direct measurement with an Atlas Xenocal Irradiance Sensor (fixed position). Lamp output stabilized after 45 min at 765 W/m2 (R2 ) 0.95). The lamp was allowed to warm for a minimum of 90 min before each experiment. Photolyses. Solar simulation was performed using a Suntest XLS+ Solar Simulator (2200 W Xe vapor lamp) manufactured by Atlas Material Testing Solutions. Light intensity was set to 765 W/m2 (300-800 nm). Screw top borosilicate vials (2 mL) were used as photoreactors. Two 14.0 cm × 27.0 cm steel trays were used to hold the vials. Each tray was designed to accommodate 92 vials, for continuous irradiation of 184 samples at a given time, and was placed on the illuminated surface during experimentation. Vials were distributed randomly in each tray to avoid spatial bias from irregularities in the light field. Sample chamber temperature was controlled by an Atlas Suncool unit. Black standard surface temperature was 33-35 °C with chamber air temperature kept at 26 °C ( 2 °C. Five vials containing actinometer solution (45) were randomly distributed among the set of 78 samples on each tray. All vials were airtight and perpendicular to the incident radiation. All were refrigerated and stored in the dark before and after irradiation. After photolysis experiments were complete, 3718
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 12, 2006
FIGURE 1. Suwannee River DOM photobleached the most rapidly at 350 nm. Absorbance was measured at t ) 0, 5, 10, 20, 30, 40, and 50 h and is shown normalized against the absorbance at t ) 0. Experimental conditions were as follows: 30.00 mg/L [SRDOM]o; 2.00 µM Fe(III); 29.50 µM NO3-; 17.50 ppt salinity; 26 °C ( 0.5; and light intensity of 765 W/m2 summed over the range 290-800 nm. reactor contents were transferred to 96-well microplates for spectrophotometric analysis. No SRDOM-containing solutions (standard or irradiated sample) were stored for more than 2 days pre- or postirradiation. No significant changes in the optical properties of stored SRDOM-containing solutions were observed over this time scale. The borosilicate vials used during photolysis had essentially no absorbance above 300 nm but did weakly absorb from 290-300 nm (A ) 0.10 at 290 nm). Analytical Measurements. All measurements were taken using a SpectraMax M2 UV-vis Scan Microplate Reader (Molecular Devices Corporation) for absorbance measurements and an FLx800 Microplate Reader (Bio-Tek Instruments) for fluorescence measurements. All absorbance and fluorescence measurement were done in a 96-well solid glass microplate. The same solid glass microplate was used throughout to eliminate plate-to-plate variation. Aliquots (400 µL) of all samples were withdrawn and loaded into the corresponding well on the microplate for fluorescence or absorbance measurements. Average integrated fluorescence intensity was measured for each actinometry sample at an excitation wavelength of 340 nm/30 nm and an emission wavelength of 420 nm/50 nm. The absorption spectrum (5 nm resolution) was measured for every sample over the range 290-500 nm. The observed response was the experimental rate constant (kobs) of SRDOM photobleaching as a function of all components for absorbance measurements at λ ) x nm over time (h). Slopes (-kobs) were generated from a plot of the normalized absorbance values (At/Ao) for each sample at λ ) x nm versus time (hours).
Results and Discussion Photobleaching. The absorbance of SRDOM with time was measured under all conditions, and photobleaching occurred broadly across the measured range of 290-500 nm (Figure 1). The most rapid rate of photobleaching occurred in a band centered at 350 nm. Rapid photobleaching in this portion of the SRDOM absorption spectrum is consistent with that reported for DOM with widely varying geographic origins and is usually attributed to the loss of quinones, aromatics, or charge-transfer complexes (1, 4, 46, 47). Bleaching appeared to be first order in SRDOM for any given single set of experimental conditions, in accordance with the observations of other researchers (Table 2) (10, 14, 15). The contributions of the four factors Fe(III), NO3-, SRDOM, and salinity (x1, x2, x3, and x4, respectively) to kobs were evaluated by statistical analysis of a model of the system. Initially, a full second-order model (eq 1) was fitted to the data, including an intercept (β0), linear terms (β1x1, β2x2, β3x3, β4x4), squared terms (β11x12, β22x22, β33x32, β44x42), and cross-
TABLE 2. Experimental Conditions (Factor Levels) and Corresponding kobs for Photobleaching at 350 nm run
SRDOM (mg/L)
salinity (ppt)
Fe(III) (µM)
NO3(µM)
(kobs) × 103 (h-1)
( n)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
7.50 7.50 7.50 7.50 22.50 22.50 22.50 22.50 7.50 7.50 7.50 7.50 22.50 22.50 22.50 22.50 15.00 15.00 15.00 15.00 0.00 30.00 15.00 15.00 15.00
8.75 8.75 8.75 8.75 8.75 8.75 8.75 8.75 26.25 26.25 26.25 26.25 26.25 26.25 26.25 26.25 17.50 17.50 17.50 17.50 17.50 17.50 0.00 35.00 17.50
1.02 3.06 1.02 3.06 1.02 3.06 1.02 3.06 1.02 3.06 1.02 3.06 1.02 3.06 1.02 3.06 0.00 4.08 2.04 2.04 2.04 2.04 2.04 2.04 2.04
14.70 14.70 44.20 44.20 14.70 14.70 44.20 44.20 14.70 14.70 44.20 44.20 14.70 14.70 44.20 44.20 29.45 29.45 0.00 58.95 29.45 29.45 29.45 29.45 29.45
8.43 ( 0.63 7.58 ( 0.57 8.63 ( 0.65 7.82 ( 0.59 11.80 ( 0.89 12.44 ( 0.93 10.88 ( 0.82 12.05 ( 0.90 9.02 ( 0.68 8.22 ( 0.62 8.92 ( 0.67 8.95 ( 0.67 12.73 ( 0.95 12.31 ( 0.92 12.74 ( 0.96 12.66 ( 0.95 11.13 ( 0.83 11.09 ( 0.83 10.99 ( 0.82 10.84 ( 0.81 1.39 ( 0.10 12.87 ( 0.97 9.21 ( 0.69 11.40 ( 0.85 11.16 ( 0.84
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 6
TABLE 3. β Values and Corresponding t-Test Comparisons for the Full Factor Model, at a 95% Level of Confidence parameter
factor effect
β0 β1 β2 β3a β4 β11 β22 β33a β44 β12 β13 β14 β23 β24 β34
y-intercept Fe(III) NO3SRDOM salinity (S) Fe(III)-Fe(III) NO3--NO3SRDOM-SRDOM S-S Fe(III)-NO3Fe(III)-SRDOM Fe(III)-S NO3--SRDOM NO3--S SRDOM-S
a
(βx est) × 104 (SE) × 104 |tcalc| prob > |t| 114.00 6.08 2.66 28.00 2.06 3.24 0.16 -9.30 -1.40 2.45 5.28 -2.00 -1.30 1.16 0.38
6.87 15.60 3.75 3.75 3.75 6.92 1.36 1.36 1.36 3.20 3.20 3.20 1.42 1.42 1.42
0.39 0.70 0.71 0.48 7.46 |t|
y-intercept SRDOM SRDOM-SRDOM
110 22.1 -9.16
16.1 1.23 1.18
18.0 -7.73
