Environ. Sci. Technol. 2000, 34, 2231-2236
Photolysis of the Resin Acid Dehydroabietic Acid in Water N I N A S . C O R I N , * ,† PETER H. BACKLUND,‡ AND MAARET A. M. KULOVAARA§ Department of Organic Chemistry, Åbo Akademi University, Biskopsgatan 8, FIN-20500 Turku, Finland, Turku Regional Institute of Occupational Health, Tavastgatan 10, FIN-20500 Turku, Finland, and Pirkanmaa Regional Environment Centre, P.O. Box 297, FIN-33101 Tampere, Finland
The effects of UV254-radiation and artificial solar radiation on the degradation of dehydroabietic acid (DHAA) in humic water and in humus-free control water were examined. The reaction rates were determined, the degradation products were tentatively identified, and the toxicity changes of the solutions were monitored. Our results demonstrate that the presence of dissolved organic matter (DOM) in water affects the light-induced degradation rate of DHAA. In the UV-experiments, the degradation was substantially slower in humic water than in humus-free control water, whereas the degradation rate was accelerated by the presence of DOM in the simulated sunlight-experiments. These differences are obviously due to different reaction pathways in the experiments. Irradiation of the aqueous DHAA solutions gave rise to a great number of degradation products of which e.g. 7-oxodehydroabietic acid and 7-oxodehydroabietin were formed in high amounts. During photolysis of DHAA in humic water, decarboxylation of DHAA to dehydroabietin (18-norabieta-8,11,13-triene) seemed to be one of the main reactions. The bacterial toxicity of the aqueous DHAA solutions decreased with increasing irradiation time. Consequently, the photolysis of DHAA did not generate any notable amounts of toxic intermediates, or the intermediates formed were rapidly further degraded into compounds of lower toxicity than the parent compound.
Introduction Resin acids are weak hydrophobic organic acids that occur naturally in the resin of wood and tree bark. They are released from the wood during chemical and mechanical pulping processes and are among the most common components found in effluents from pulp and paper mills. The most important resin acids are pimaric, sandaracopimaric, isopimaric, levopimaric, palustric, dehydroabietic (DHAA), abietic, and neoabietic acid. Of these compounds, DHAA is the most abundant and resistant one. Resin acids are major contributors to the toxicity of effluents from pulp and paper mills (1). The acute toxicity or LC50 (median lethal concentration) values for individual resin acids in rainbow trout range from 0.4 to 1.7 mg/L (2), and DHAA has a sublethal toxicity of only * Corresponding author phone: +358-2-215 4501; fax: +358-2215 4866; e-mail:
[email protected]. † Åbo Akademi University. ‡ Turku Regional Institute of Occupational Health. § Pirkanmaa Regional Environment Centre. 10.1021/es9910816 CCC: $19.00 Published on Web 04/29/2000
2000 American Chemical Society
20 µg/L (3). Furthermore, resin acids accumulate in freshwater mussels (4) and in fish tissues (3, 5). Resin acids are highly resistant to chemical degradation, but modern activated sludge systems (6) and aerated stabilization basins (7) can remove more than 90% of the resin acids from the effluents. Kaplin et al. (8) found, for example, that the concentration of resin acids in a biologically treated pulp mill effluent was below 150 µg/L. Nevertheless, a considerable amount of resin acids is released into the surrounding waters. Although resin acids are biodegradable, they are quite persistent in the environment. DHAA has an apparent half-life of about 0.12 years in water (9) and 30 years in sediments (10). Because of their ability to adsorb onto suspended solids in the effluents a wide variety of resin acids have been detected in sediments close to pulp and paper mills (11). Tavendale et al. (12) further showed that resin acid-based neutrals, such as dehydroabietin, fichtelite, and retene, are formed through anaerobic bacterial processes in sediments enriched in resin acids. These compounds are more lipophilic than the parent resin acids and therefore bioaccumulate in organisms to a larger extent. Retene (1methyl-7-isopropyl phenanthrene) is a polycyclic aromatic hydrocarbon (PAH) compound, which is widely recognized to be of natural origin and has been shown to accumulate in fish tissues and further to be a strong inducer of mixed oxidase function (MFO) in fish (13). Humic substances are high-molecular-weight (HMW), recalcitrant, and yellow-brown colored organic compounds that are found wherever plant and animal material are degraded. The major fraction of the dissolved organic matter (DOM) in surface waters consists of humic substances. They may account for up to 90% of the dissolved organic carbon (DOC) content (14-16). Humic substances are the major chromophores in waters and therefore play a dominant role in various photochemical processes in lakes and rivers. When humic substances absorb UV or solar radiation reactive oxygen intermediates (17, 18), so-called photoreactants are formed. These reactive intermediates might in turn accelerate the light-induced transformation of organic compounds in natural waters. A dissolved organic pollutant may be degraded either by direct or indirect photolysis. Direct photolysis occurs when a given pollutant absorbs radiation and as a consequence undergoes transformation. Indirect or sensitized photolysis occurs either by direct energy transfer from excited humic substances to the pollutant or by the effects of other reactive intermediates. The anticipated increase in the global UV-radiation and the growing interest in utilizing UV-radiation in water treatment processes require a better understanding of UV and sunlight mediated chemical processes taking place in natural humic surface waters. The purpose of the present study was to determine the effects of DOM on the lightinduced degradation of DHAA in water and to identify the major degradation products. The photolysis of DHAA was conducted in natural humic freshwater and in humus-free control water by using artificial sunlight or UV254-radiation. Moreover, the bacterial toxicity of the aqueous DHAA solutions was determined after specific irradiation times.
Experimental Section Water Samples and Chemicals. Humic surface water (DOC ) 25.5 mg/L, pH 6.2) was collected in November 1997 from Lake Savoja¨rvi in southwestern Finland. The lake is situated in a marsh region and is relatively unaffected by municipal or industrial activities. The water was immediately filtered with 0.45 µm (UV-experiments) or 0.22 µm (simulated VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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sunlight-experiments) membrane filters (Millipore HATF) and stored at +4 °C in the dark before use. Milli-Q water was used as a solvent for control samples. All chemicals used in the experiment were of p.a. quality or of higher purity. Dehydroabietic acid of 99% purity was received from Helix Biotech (Canada). A stock solution of DHAA (8.18 mg/mL) was prepared in ethanol, and 250 µL of the solution was added to 1 L filtered humic water or to 1 L humus-free control water, resulting in an overall DHAA concentration of 2 mg/L (6.7 µM). The prepared water samples were then stirred for 15 min or 24 h in the dark at room temperature before the irradiation. A standard stock solution of heptadecanoic acid (8.08 mg/mL) was prepared in freshly distilled methyl tert-butyl ether (MTBE, LabScan). Bis(trimethylsilyl)trifluoroacetamid (BSTFA, Merck) was purchased from Merck and pyridine was obtained from Riedelde Hae¨n. Irradiation of Water Samples. The prepared water samples were exposed to either ultraviolet radiation (254 nm, UV254) or simulated sunlight (300-800 nm), with a delay of 15 min or 24 h after the spiking. The aqueous DHAA solutions were exposed to UV254-radiation, in a quartz glass tube (5.0 cm o.d. × 43.0 cm, with a glass stopper), by using six 15-W low-pressure mercury discharge UV-lamps (Philips TUV). The lamps were installed vertically in a reflector and placed at a distance of 30 cm from the center of the sample tube. The Hg lamps emit almost all of their energy as a sharp spectral line at 253.7 nm, and according to the manufacturer, the radiation intensity of one lamp at a distance of 1 m is 42 µW/cm2. During the irradiation the samples were stirred by a magnet. For the quantification of DHAA and for the analyses of degradation products 50-mL samples were taken after specific irradiation times. The irradiation times were 0-420 min for DHAA in humic water and 0-150 min for DHAA in control water. The aqueous solutions of DHAA were exposed to simulated sunlight in a Heraeus Xenotest 150 S apparatus equipped with a 1.3-kW xenon burner and special glass filters. The emitted radiation has a spectral distribution similar to that of natural sunlight. According to the manufacturer, the lamp has an optimum effect of approximately 0.1 W/m2 at 290300 nm, 146 W/m2 at 300-400 nm, and 1220 W/m2 at 400800 nm. By comparison, the intensity of the solar radiation in Turku (Finland) on a sunny summer day seldom exceeds 700 W/m2 (Process Design Laboratory, Åbo Akademi University). The water samples were irradiated in test tubes, 10 × 20 mL (Duran, 18 mm o.d. × 16 cm, with screw caps), for up to 8 h at a temperature of 33 ( 2 °C. The test tubes were placed at a distance of 2.5 cm from the filters on Teflon holders, which rotated around the burner and turned 180° during every revolution. The glass walls of the test tubes absorb light of wavelengths below approximately 310 nm. For the quantification of DHAA and for the analyses of degradation products aliquots of 40 mL were withdrawn after specific irradiation times. Liquid-Liquid Extraction. Aliquots of 50 mL (UV254experiment) and 40 mL (simulated sunlight-experiment) of the treated water samples were extracted at ambient pH 6.2 with four portions (15 mL + 10 mL + 10 mL + 10 mL) of MTBE. A known amount of the standard stock solution of heptadecanoic acid was added as an internal standard together with the first portion of the solvent. The organic extracts were combined, and the residual water was frozen out at -20 °C overnight and then concentrated by rotary evaporation under reduced pressure to a volume of approximately 5 mL. The extracts were transferred to glass vials and further concentrated to approximately 0.5 mL with a stream of pure nitrogen and derivatized by silylation before GC/MSD analysis. The samples were silylated by adding 100 µL of BSTFA and a drop of pyridine to the sample. The vials 2232
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were then sealed and kept at 60 °C for 30 min. After the derivatization the reaction mixtures were cooled, concentrated with a gentle stream of pure nitrogen, and analyzed immediately by GC/MSD. Analytical Procedures. DOC was measured with a Shimadzu Total Carbon 5050 Analyzer equipped with an automatic sample injector (Shimadzu ASI 5000). The UVabsorbance was determined with a Shimadzu UV-160 UVvis spectrophotometer. The derivatized samples were analyzed with a Hewlett-Packard 5971A mass selective detector equipped with a Hewlett-Packard 5890 Series II gas chromatograph (GC/MSD). The GC peaks were separated on a HP 1 capillary column of 25 m length and 0.2 mm i.d. with a stationary phase film thickness of 0.33 µm. The injector and the interface temperature were set at 280 °C and 300 °C, respectively. The GC oven temperature was held at 100 °C for 2 min and then raised to 300 °C (4 min) at a rate of 8 °C/min. DHAA was quantified by determining the area response of DHAA relative to that of the internal standard heptadecanoic acid. Determination of Response Factors and Extraction Efficiency. The response factors were determined by mixing known amounts of the internal standard heptadecanoic acid and DHAA in 0.5 mL of MTBE, and the sample was derivatized and analyzed as described above. Response factors were calculated on the basis of the known concentrations and the analysis results were corrected for the response. To determine the extraction recoveries, five or six parallel samples of DHAA in filtered humic water and in humus-free control water were extracted as described above, 15 min or 24 h after the spiking. Toxicity Test. Toxicity was measured by using a BioTox luminescence assay. This assay measures the reduction in the natural light emission of the marine organism Vibrio fischeri. The bacteria produce light as a part of their natural metabolism, and when toxins interfere with these metabolic processes a reduction of the light emission occurs. Solutions of DHAA were prepared by adding 187 µL of the DHAA stock solution to 500 mL of humic water or to 500 mL of humus-free control water. To improve the aqueous solubility of DHAA the pHs of the solutions were adjusted to 7 with 0.05 M NaOH. The final DHAA concentration was 3 mg/L, which is below the reported water solubility of 4.9 mg/L for DHAA (19). The solutions were stirred by a magnet for 15 min and subsequently exposed to UV254-radiation or simulated sunlight. Test tubes, used in the simulated sunlightexperiments, were sterilized by autoclaving (121 °C, 15 min) before use. In addition, water samples from the UV254experiments were withdrawn in sterilized test tubes. The toxicity was measured within 24 h after the irradiation. Solid NaCl was added to the samples to give a 2% salt concentration, and the pHs of the samples were checked and if necessary adjusted to 7 immediately before the toxicity measurements. A volume of 200 µL of the Vibrio fischeri suspension was added to 800 µL of the treated water sample and then incubated at 15 °C for 30 min. The reduction in light emission was monitored on a BioOrbit 1253 Luminometer. The results are reported as a mean value of three replicates measured for each sample.
Results and Discussion Photolysis of DHAA. The extraction efficiencies were tested after an equilibrium time of 15 min or 24 h after the spiking, and the recoveries of DHAA from humic water were 96 ( 2% (n ) 6) and 97 ( 2% (n ) 5), respectively. The recovery of DHAA from control water 15 min after the spiking was 99 ( 3% (n ) 6). Since it is known that microorganisms may degrade DHAA in water (20), we also determined the extraction recovery of DHAA from water solutions kept in the dark for up to 8 h at a slightly elevated temperature (33
TABLE 1. Pseudo-First-Order Photodegradation Kinetics of DHAA in Humic Watera and in Humus-Free Control Water sample
n
R2
intercept
slope (min-1)
humic water (15 min) humic water (24 h) control water
26 22 27
0.983 0.982 0.996
UV254-Irradiation 0.19 -0.007 0.14 -0.007 0.01 -0.020
humic water (15 min) humic water (24 h) control water
25 18 20
0.981 0.982 0.946
Simulated Sunlight-Irradiation 0.19 -0.011 0.30 -0.010 0.15 -0.003
a
15 min and 24 h contact time before irradiation.
b
95% C.I.b (min-1)
half-life (min)
-0.008 to -0.006 -0.008 to -0.006 -0.0204 to -0.0203
99 99 35
-0.012 to -0.010 -0.011 to -0.009 -0.004 to -0.003
63 69 231
95% confidence interval (C.I.) for the slope of the regression line.
FIGURE 1. Pseudo-first-order degradation plot for UV254-irradiated solutions of DHAA in humic water (15 min and 24 h contact time) and in humus-free control water. Each data point represents an average of two or three measurements; standard deviations are given as error bars, and the rate coefficients for the degradations are presented.
FIGURE 2. Pseudo-first-order degradation plot for simulated sunlight (300-800 nm) treated solutions of DHAA in humic water (15 min and 24 h contact time) and in humus-free control water. Each data point represents an average of two or three measurements; standard deviations are given as error bars, and the rate coefficients for the degradations are presented.
°C) in the Xenotest apparatus. The obtained recoveries were 94 ( 4% (n ) 4) from humic water and 96 ( 4% (n ) 4) from control water, i.e., DHAA was not noteworthy degraded in the absence of light. DHAA was degraded both by UV254-radiation and simulated sunlight. The degradation of DHAA by UV254-radiation was substantially slower in humic water than in control water, whereas the opposite effect was observed in the simulated sunlight-experiments. UV254-irradiation for 120 min degraded 39% and 48% of the DHAA in humic water (15 min and 24 h contact time before irradiation), whereas more than 90% of the DHAA was degraded in control water. Simulated sunlight exposure for 120 min resulted in a 53% and 64% (15 min and 24 h contact time, respectively) decline in the DHAA concentration in humic water and a 33% decline in control water. Each irradiation series was repeated two or three times, and the recovery of DHAA after specific irradiation times is reported as a mean value of the determinations. The results are presented as natural logarithmic values of the DHAA recovery versus irradiation time (Figures 1 and 2). All experiments gave linear plots, indicating that the photodegradation of DHAA in humic and in control water follows pseudo-first-order kinetics. The regression data, the 95% confidence intervals for the slopes of the regression lines,
and the pseudo-first-order half-lives are presented in Table 1. The fact that all the regression lines do not pass origin is probably due to experimental inaccuracy, and the regression lines are not forced to pass through zero. The degradation rate constants were calculated and resulted in the following data: the rate constants of DHAA in humic water exposed UV ) 0.12 × 10-3 s-1 and kUV to UV254-radiation were k15min 24h ) -3 -1 0.12 × 10 s (15 min and 24 h contact time before irradiation). In the simulated sunlight-experiments, the a.sun corresponding rate constants were k15min ) 0.18 × 10-3 s-1 a.sun -3 -1 and k24h ) 0.17 × 10 s with 15 min and 24 h contact time before the irradiation, respectively. Consequently, the contact times (15 min and 24 h before irradiation) between DOM and DHAA did not affect the magnitudes of the rate constants, indicating that no strong associates were formed between DOM and DHAA. These results agree with the findings of Kukkonen and Oikari (21), who determined the binding coefficient (Kp) between DOM and DHAA and received Kp values as low as ∼500 L/kg for DHAA in natural humic freshwater. The degradation rate constants for DHAA in control water exposed to UV-radiation or simulated sunlight were kUV ) 0.33 ×10-3 s-1 and ksun ) 0.06 × 10-3 s-1, respectively. VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. GC/MS Characteristics of the Photoproducts Tentatively Identified in Aqueous Solutions of DHAA Exposed to UV254-Radiation or Simulated Sunlight (300-800 nm) compd
RT (min)
parent compd (mol wt)
1 2 2a 3 4 5 5a 6 6a 7 8 9
16.9 18.6 19.6 19.5 19.8 21.6 22.1 22.5 22.7 23.1 23.3 25.0
256 272 272 270 272 286 286 388 388 380 388 387
TMS derivative (mol wt) 344 344 344 358 358 460 460 386 460 474
The effects of UV-radiation on DOM have been studied in many recent papers, and short reviews are given in the papers by Frimmel (22, 23). Previous reports have, for example, shown that the HMW material is partially mineralized to carbon dioxide and water and partially degraded into compounds of smaller molecular size including a lower degree of conjugation. In the present study, the DOC content was decreased by 15% and 24% after 120 and 420 min of UV254-irradiation, respectively. In the simulated sunlightexperiments, the total loss of carbon (3%) was negligible and probably due to inaccuracy in the DOC-measurements. The absorbance at 254 nm (A254) declined by 8% and 19% after 120 and 420 min of UV254-irradiation, respectively. The corresponding decreases in the simulated sunlight-experiments were 2% and 7%. Conjugated double bonds in the DOM and free electron pairs on heteroatoms absorb radiation at 254 nm and a decrease in the A254 thus indicates that some of these structures are destroyed. Our results reveal that the presence of DOM in water affects the light-induced degradation of DHAA. The degradation of DHAA by UV254-radiation was retarded in the presence of DOM (Figure 1). This observed retardation can be explained either by a complex formation between DOM and DHAA or by a competition between DOM and DHAA for available photons. Humic water contains DOM, iron colloids, and various ions and salts that also absorb UV-radiation, and since no associates seemed to be formed between DOM and DHAA, the more likely explanation for the retarded photolysis rate in humic water is a competition process. The absorption spectrum of DHAA (in ethanol) shows mainly one maximum at λ ) 270 nm. DHAA, thus, absorbs radiation mainly in the UV-region, which implies that UV254-radiation is responsible for the direct photolysis of DHAA in control water. The slower photolysis rates of pesticides in humic water compared to those in distilled water have also been explained by the so-called optical filter effect of humic substances (24). The presence of natural DOM had an accelerating effect on the degradation of DHAA by simulated sunlight (Figure 2). This rate enhancement indicates that the production of reactive intermediates by DHAA itself is less important than the light-induced oxidant production by DOM. Hence, when humic solutions of DHAA are exposed to simulated sunlight, indirect photolytic processes will be the dominating ones. Similar differences in the photolysis rates of organic pollutants in humus-free control water and in humic water have also been demonstrated in previous studies (e.g. refs 25-27). Photoproducts. The degradation products formed in the aqueous DHAA solutions were tentatively identified after 3 and 6 h exposure to artificial solar- or UV254-radiation. Because of the rapid UV-photolysis of DHAA in control water, the degradation products formed in the UV-treated control water 2234
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major fragments m/z (%) 256 (26) 344 (5) 344 (10) 270 (62) 329 (100) 343 (100) 343 (100) 460 (6) 445 (39) 386 (23) 445 (100) 459 (100)
185 (64) 254 (30) 254 (61) 255 (100) 238 (6) 327 (6) 327 (8) 445 (6) 417 (27) 268 (85) 327 (6) 285 (19)
159 (100) 239 (100) 239 (56) 173 (73) 132 (21) 207 (8) 254 (4) 234 (31) 237 (80) 253 (100) 237 (3) 207 (23)
117 (48) 157 (28) 157 (100) 143 (54) 117 (25) 117 (8) 185 (6) 191 (100) 191 (48) 187 (29) 143 (4) 147 (11)
FIGURE 3. Total ion chromatogram (TIC) of degradation products formed after a 3-h UV254-irradiation of DHAA in humic water. Heptadecanoic acid was used as internal standard (ISTD, 50 µg/L). were identified after 75 and 150 min of irradiation, respectively. Figure 3 shows a total ion chromatogram (TIC) of the degradation products formed after 3-h UV254-irradiation of DHAA in humic water. The degradation products were identified by interpreting the recorded mass spectra and by comparing with spectra previously published. Nine compounds, including three pairs of isomers, were identified in the treated water samples. The mass spectra of the unlabeled peaks in Figure 3 were not characteristic enough to enable us to propose the structures. The fragmentation patterns of the silylated compounds were found to be quite similar to those of the corresponding methylated esters elucidated by previous workers (e.g. refs 28 and 29). The aromatic DHAA with an abietane skeleton shows a characteristic mass spectrum with only little fragmentation and only one intense fragment at m/z 239. This fragment results from the combined loss of methyl, trimethylsilyl, and formate radicals. The loss of this fragment (M+ - 133) is favored by many resin acids and often results in the base peak. The GC/MS characteristics and the structures of the photoproducts identified are presented in Table 2 and in Figure 4, respectively. Figure 4 shows that mainly two types of degradation products are formed during photolysis of DHAA. The first group of compounds (1-5) consists of dehydroabietin (18-norabieta-8,11,13-triene, 1) and various oxidized forms of this compound. DHAA is first decarboxylated to dehydroabietin which is further oxidized to compounds 2-5. Of these products, the most abundant ones were 7-hydroxydehydroabietin (2, 2a) in the 3-h irradiated samples and 7-oxodehydroabietin (3) in the 6-h irradiated samples. The neutral compound dehydroabietin was generated in all samples except for in UV254-treated control water (Table 3). The second group of compounds (6-9) consists of various forms of oxidized DHAA. Of these
identified methyl 7-oxodehydroabietic acid as the main degradation product. Previous studies have shown (12, 30-31) that retene is formed through a decarboxylation followed by an aromatization of the precursors DHAA and abietic acid in the sediments. Moreover, Bouloubassi and Saliot (30) reported that a large fraction of retene is formed also in the water phase before the deposition of the precursors. One major process during the light-induced degradation of DHAA in humic water was a decarboxylation of DHAA to dehydroabietin and a subsequent oxidation of this neutral compound. Since dehydroabietin is a key intermediate in the transformation of DHAA to retene, the TICs were also screened for other neutral compounds such as fichtelite, tetrahydroretene, and retene, but none of these compounds could be found.
FIGURE 4. Structures of dehydroabietic acid (DHAA) and of the degradation products (1-9) tentatively identified in aqueous DHAA solutions exposed to UV254-radiation or simulated sunlight (300800 nm).
TABLE 3. Distribution of Degradation Products Identified in Aqueous Solutions of DHAA Exposed to UV254-Radiation or Simulated Sunlight (300-800 nm) humic water
control water
UV254radiation
simulated sunlight
compd
3h
6h
3h
6h
75 min
2.5 h
3h
6h
1 2 2a 3 4 5 5a 6 6a 7 8 9
+ + + + + + + + + + + +
+ + + + + + + + + -
+ + + + + + + + + + +
+ + + + + + +
+ + + + + +
-
+ + + + + + + + -
+ + + + + -
UV254radiation
simulated sunlight
compounds, 7-hydroxydehydroabietic acid (6, 6a) and 7-oxodehydroabietic acid (7) were the most abundant ones. Similar oxidized forms of DHAA were identified in a previous study by Gigante et al. (28), who treated a 0.2 M solution of methyl dehydroabietate in butanol with UV-radiation and
The distribution pattern and the total number of degradation products differed between the various water samples. In general, the number of degradation products was larger in humic water than in control water (Table 3). Again, this might be due to the sensitizer effect of DOM in humic water. Moreover, the total number of photoproducts was considerably higher in control water exposed to artificial solar radiation than in UV254-treated control water. In all cases, the amount of degradation products seemed to increase when short irradiation times were used but decline with prolonged irradiation. The photoproducts were probably simultaneously produced and degraded as available photons were used both by the parent compound DHAA and by the degradation products formed. Consequently, the degradation of the photoproducts probably increased simultaneously as the concentration of DHAA decreased. All the photoproducts identified in the 75-min UV254-treated control water were degraded after 150 min of irradiation, and no other degradation products were detected. Accordingly, the intense UV254radiation used in the present study completely degraded DHAA and its photoproducts in the absence of DOM. We expected that photolysis of DHAA would produce compounds with several hydroxy groups, and an extraction of the treated water samples at lower pH would therefore be appropriate. One series of UV-irradiated humic water samples was also extracted at pH 2, but no other degradation products than the previous ones were detected (results not shown). Toxicity Measurements. The toxicity changes during irradiation of DHAA dissolved in humic water and in humusfree control water were monitored by using the BioTox luminescence assay. The concentrations of the aqueous DHAA solutions (3 mg/L at pH 7) were low due to solubility problems, and the volume of the bacteria suspension was kept small in order to prevent any further dilution. Together these facts made the toxicity test very sensitive for pH variations, and it was important to adjust the pH to 7 immediately before the measurements. Because of the low initial DHAA concentration it was not possible to determine an EC50 value for DHAA in the waters with this method. It has also been shown (32) that the bacterial toxicity of DHAA (15-min EC50 ) 11 mg/L) is much lower than its toxicity (96-h LC50 ) 1.1 mg/L) for rainbow trout (2). Since the toxicity values measured with different test organisms may differ, it is important to use several test organisms to receive reliable results. The rapidity and low costs of the luminescence assay makes it a very attractive method, and it is one of the most used toxicity tests. Another toxicity test, which is commonly used, measures the growth of the green algae Raphidocelis subcapitata (former Selenastrum capricornutum). The toxicity of DHAA to Raphidocelis subcapitata was measured according to a standardized test protocol (33), but because of the low concentrations of DHAA the test appeared to be too insensitive to be used in this study. Hence, the toxicity values were determined only by using the luminescence assay. VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 4. Relative Toxicitya Values in Aqueous Solutions of DHAA Exposed to UV254-Radiation or Simulated Sunlight (300-800 nm) DHAA in DHAA in control water humic water irr time humic water (min) cDHAA (mg/L) rel tox rel tox cDHAA (mg/L) rel toxb 0 30 150 360
3.0 2.0 0.4
0 120 360
3.0 2.2 0.7
UV254-Irradiation 1.0 1.0 0.74 0.43 2.1 2.0
3.0
1.0
2.2 1.0
0.74 0.89
Simulated Sunlight-Irradiation 1.0 1.0 3.0 0.70 1.1 2.1 0.45 1.6 0.1
1.0 0.95 0.62
aRelative toxicity (rel tox): The toxicity is defined relative to the original sample (before irradiation) as having a relative toxicity of 1. b Values corrected with respect to the relative toxicity in unspiked humic water.
The bacterial light emission in unspiked humic water, determined as a background control test, decreased by 9%. Consequently, the toxicity data determined for DHAA in humic water had to be corrected by this background value. According to the manufacturer of the luminescence assay, the color of the humic water measured as absorbance at 460 nm (A460) should not exceed 0.2. The color of the humic water used in this study was 0.06 (A460), which is below the reported limit value. The fact that the concentrations of both metal ions and DOM in Lake Savoja¨rvi are relatively high might explain the decreased light emission in the unspiked humic water. The bacterial light emission in the unexposed DHAA solutions were inhibited by 36 ( 1% and 23 ( 2% in humic water and in humus-free control water, respectively. After the background correction, no obvious differences in the toxicity values between DHAA in humic water (27%) and in humus-free control water (23%) could be observed. Furthermore, the results in Table 4 show that the relative toxicity values of unspiked humic water increased with prolonged irradiation time. This increase in toxicity could be explained not only by the formation of toxic oxygen species but also by the release of metal ions from the bulk organic material during the irradiation. After 150-min UV254-irradiation, the toxicity of DHAA in humic water and in control water was decreased by 26% and 60%, respectively. The corresponding decreases following 120-min exposure to artificial solar radiation were 5% (humic water) and 30% (control water), respectively. In all cases, except for DHAA in humic water exposed to UV254-radiation, the toxicity values correlated well with the decreasing DHAAconcentration. The decreased toxicity might therefore be explained by a diminished concentration of DHAA and/or by a formation of compounds of lower toxicity than the parent compound. Hence, our results reveal that the presence of DOM do not affect the bacterial toxicity of DHAA in water solutions and further that the toxicity decreased with increasing irradiation time. The initial increased toxicity of DHAA in humic water was due to the background inhibition of the light emission in unspiked humic water. In conclusion, our results demonstrate that light-induced degradation is an important removal pathway of DHAA from aquatic environments. When exposed to artificial solar radiation the degradation of DHAA proceeds faster in humic freshwaters than in clear waters. The degradation of DHAA is, on the other hand, retarded by the presence of DOM when
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exposed to UV254-radiation. In water treatment processes a higher intensity of the UV-radiation will be needed for the removal of DHAA from humic waters than from nonhumic waters.
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Received for review September 20, 1999. Revised manuscript received February 28, 2000. Accepted March 10, 2000. ES9910816
