Environ. Sci. Technol. 1994, 28, 1389-1393
RESEARCH COMMUNICATIONS Hydroxyl Radical Mediated Degradation of Azo Dyes: Evidence for Benzene Generation Jack T. Spadaro,t Lome Isabelle,* and V. Renganathan'vtp*
Department of Chemistry, Biochemistry, and Molecular Biology, and Department of Environmental Science and Engineering, Oregon Graduate Institute of Science & Technology, Portland, Oregon 9729 1-1000 Introduction Azo dyes constitute the largest class of dyes used in industry (1). Approximately 10 000 different dyes and pigments are used industrially, and over 7 X lo6 of these dyes are produced annually worldwide (1). Textile industries are the largest consumers of dye stuffs, and it is estimated that 10-15% of the dye is lost during the dyeing process and is released as effluent (2). Azo dyes are resistant to aerobic degradation (3,4);however, under anaerobic conditions, they can be reduced to potentially carcinogenic aromatic amines (5-9). Also, in mammals, cytochrome P-450 reductase and intestinal microflora reduce azo linkages to generate aromatic amines in uiuo (10,ll). Popular treatment methods for eliminating dyes from the waste stream include flocculation with lime, activated charcoal adsorption, and biotreatment (12). Lime treatment and charcoal adsorption generate solid wastes, which require costly disposal methods. Biotreatment processes rely on indigenous soil microorganisms to degrade dye compounds. Since the synthetic dyes are resistant to aerobic biodegradation, this process is likely to be inefficient. Thus, there is a need for developing treatment technologies that are more effective in eliminating dyes from the waste stream at its source. Advanced oxidation processes refer to the processes in which the highly reactive hydroxyl radicals ('OH) are used for degrading on organic pollutant, usually to C02. Reaction of *OHwith aromatic compounds occurs almost at the diffusion-controlled rate (13). The initial products of these reactions are the corresponding mono- and dihydroxylated derivatives (14). Continued reaction of the hydroxylated compounds with 'OH leads to aromatic ring cleavage. Products arising from ring cleavage are subsequently oxidized to COz. Since 'OH is relatively nonspecific in its reactions, it is useful for degrading a variety of environmental pollutants including chlorobenzene (15), polychlorobiphenyls (16),chlorophenols ( 1 3, dichlorophenoxyacetic acid (18),nitrobenzenes (19),nitrophenols (201,perchloroethylene (21),and formaldehyde (22). Chemical and photochemical reactions are used for 'OH generation. In the chemical process, the reaction of a ferrous or a ferric salt with H202 produces 'OH as shown ~
~
~~
* To whom correspondence should be addressed at Department
of Chemistry, Biochemistry,and Molecular Biology,Oregon Graduate Institute of Science & Technology, P.O. Box 91000, Portland, OR 97291-1000. Telephone: (503) 690-1134. Fax: (503) 690-1464. t Department of Chemistry, Biochemistry, and Molecular Biology. t
Department of Environmental Science and Engineering.
0013-936X/94/0928-1389$04.50/0
@ 1994 American Chemical Society
in eq 1-3 (23): Fe(I1)
+ H20, + H+
-
-
Fe(II1)
+ 'OH + H 2 0
Fe(II1) + H202 Fe(I1) + '02H + H+ Fe(II1) + 'OOH
Fe(I1) + 0, + H+
In the photochemical reaction, *OH is produced via UVH202, UV-Ti02, or UV-O3 reaction (24-26). Fe2+-H202 treatment has been suggested as an alternative method for removing dyes from industrial effluent (27-29);however, a detailed study of this process has not been performed. In this report, using 14C-labeleddyes, we demonstrate that 'OH, generated via the reaction of Fe3+and H202 at pH 2.8, partly degrade azo dyes to C02. We also present evidence for the generation of benzene and substituted benzenes from 'OH radical degradation of azo dyes with a phenylazo substitution. A probable mechanism for benzene generation is also proposed. Among the dyes used in this study, 4-phenylazoaniline is a mutagen whereas, N,N-dimethyl-4-phenylazoaniline, Disperse Yellow 3, and Solvent Yellow 14 are carcinogens ( 1 , 8 ,30). Experimental Section
Chemicals. %-Labeled azo dyes were synthesized and purified as described by Spadaro et al. (31). 4-Phenylazophenol and 4-phenylazoaniline were obtained from Fluka, Ronkonkoma, NY. Disperse Yellow 3, Solvent Yellow 14, and Disperse Orange 3 were purchased from Aldrich Chemical Co., Milwaukee, WI. N,N-Dimethyl4-phenylazoanilinewas synthesized as previously described (31). Ferric nitrate [Fe(N03)~9HzOland H202 were purchased from Sigma Chemical Co., St. Louis, MO. Degradation of W-Labeled Azo Dyes. A radiolabeled dye (50 000-72 000 cpm, 50 pM) in ethanol (250 pL) was added to deionized water containing 0.1 % Tween-80, ferric nitrate (2 mM), and nitric acid (1mM). Tween-80, a nonionic detergent, was needed to solubilize the dyes into solution. Reaction was started with the addition of H202 (75 mM final concentration). The reaction flask was sealed with a gas-exchange manifold and shaken on a rotary shaker (200 rpm) in the dark. 14C02released due to 14C-labeleddye degradation was trapped with NaOH (1N, 0.6 mL). NaOH was kept in a polycarbonate vial, which was attached to the manifold. After 10 h, shaking Environ. Sci. Technoi., Vol. 28. No. 7, 1994
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was stopped; the head space was purged with air for 15 min, and the purged air was passed through 1mL of 1N NaOH to capture untrapped 14C02. H202 (75 mM final concentration) was once again added to the reaction mixture. After replacing the CO2 trap with a fresh one, the reaction was continued for an additional 14 h. The amount of radioactivity in C02 released after 24 h was collected as described above. The amount of radioactivity in the C02 traps was determined using a liquid scintillation counter (Model LS-3133P, Beckman Instruments Inc., Fullerton, CA). Ecolite (ICN Biomedical) was used as the scintillation fluid. The sum of 14C02released after 10 and 24 h represented the total amount of dye degraded to C02 in 24 h. The quantity of water-soluble and methylene chloride-soluble products formed at the end of the 24-h reaction also was determined. Initially, the amount of radioactivity remaining in the reaction mixture was determined. Then 1 mL of the reaction mixture was extracted with methylene chloride (1 mL), and the radioactivity remaining in the aqueous phase, presumably due to water-solubleproducts, was estimated. The amount of radioactivity in the methylene chloride fraction was calculated by subtracting the radioactive counts in the aqueous phase after methylene chloride extraction from the total radioactive counts that were in solution before methylene chloride extraction. Four replicate samples were used for each azo dye degradation experiment. Determination of Total Volatile Compounds. Reaction conditions were the same as described above for the 14C-labeledazo dye degradation experiments except that H202 (75 mM) was added only once. Volatile organic components were trapped by purging the head space with air and bubbling the purged air through a 2-methoxyethanol-toluene mixture (1:1,vol/vol) containing scintillation fluors. The amount of radioactivity trapped was determined by liquid scintillation counting. Volatile organics release was determined at 1.5, 3, 4.5, and 6 h. Simultaneously, the amount of W02released was also determined as described above. Identification of Volatile Organics. The nature of volatile organics formed from the degradation of 4-phenylazophenol (I), 4-phenylazoaniline (3),N,N-dimethyl4-phenylazoaniline (4), and Solvent Yellow 14 (6) were determined. Reaction mixtures contained dye (50 pM added in 125 pL of methanol), Fe3+ (2 mM), nitric acid (1 mM), and H202 (75 mM). In addition, 4-phenylazophenol (1) and Solvent Yellow 14 (6) reactions contained 0.1% Tween-80. The total reaction volume was 25 mL, and reactions were conducted in 40-mL stoppered vials fitted with Teflon-silicone septa (Pierce, Rockford, IL). Also, the reactions were performed in the dark at room temperature for 1.5-4 h. The gas phase of the reaction mixture was analyzed by agas chromatograph (HP 5790A, Hewlett Packard Co.) coupled to a mass spectrometer (Finnigan 4000, Finnigan Associates). The GC column (Econocap Carbowax, Alltech Associates, Deerfield, IL) was maintained at an initial temperature of -50 "C, and the injector was at 200 "C. The column temperature was increased from -50 to 270 OC at the rate of 15 "C/min during analysis. HPLC and G U M S Analyses. HPLC analyses were performed using a C-18 reversed-phase column (Separations Group, Hesperia, CA) and a gradient eluent consisting of water and methanol. The methanol concentration was maintained at 20% for 2 min; then it was increased to 1390 Environ. Scl. Technol., Vol. 28, No. 7, 1994
100% in 10 min and maintained at 100% concentration for an additional 10 min. Compound elution was monitored at 254 nm. GC/MS analyses were performed at 70 eV on a VG Analytical 7070E mass spectrometer fitted with an H P 5790A GC and a fused capillary column (15 m, DB-5, J&W Science). A temperature gradient was used in the GC separations. The initial temperature was 70 "C, and it was increased to 320 "C at a rate of 10 "C/min.
Results and Discussion In this paper, degradation of specifically '4C-labeled azo dyes by 'OH, generated via the reaction between ferric nitrate and H202, at pH 2.8 was studied. Dyes used in this study are listed in Table 1. The optimal concentrations of ferric nitrate and H2Oz required for dye degradation were identified by studying the effect of Fe3+ and H202 concentrations on the phenolic ring-labeled 4-phenylazophenol (la) degradation to C02. Fe3+ at 2 mM and H202 at 75 mM concentrations provided the highest levels of 14C02from la (data not shown). These concentrations of Fe3+ and H202 were subsequently used in all the dye degradation reactions. Since many of the azo dyes investigated are water-insoluble, Tween-80 was included to solubilize them. Preliminary studies on the effect of Tween-80 on phenolic ring-labeled 4-phenylazophenol degradation to CO2 suggest that Tween-80 does not affect dye degradation significantly. All the dyes examined were totally decolorized in 24 h, indicating the loss of their chromophoric group. Degradation of dyes to CO2 ranged from 8 to 30% (Table 1). In general, high levels of 14C02 evolution were observed with phenolic or aniline ringlabeled azo dyes such as la, 3, and 4a (Table 1). About 29-63 % of the aromatic ring appears to have been degraded to water-soluble products. The initial pH of reaction was 2.8, and the final pH was 2.3. Degradation Products. Disperse Yellow 3 (3) degradation reaction, when analyzed by HPLC after 4 h, indicated the presence of two products (Rt = 3 and 13.85 min). Mass spectrum of the product with a retention time of 13.85min corresponded to that of acetanilide. MS (m/ 2 ) : 135 (46.6%); 93 (100%);77 (4.2%). Acetanilide yield was estimated to be approximately 44.7 mol % . Disperse Orange 3 (5) reaction mixture also showed the presence of two peaks (Rt = 3 and 16 min) in HPLC analysis. The product with a retention time of 16 min corresponded to nitrobenzene; GC/MS analysis of the methylene chloride extract of the reaction mixture confirmed the presence of nitrobenzene. MS (mlz): 123 (61.9%); 77 (100%); 65 (11.6%). Nitrobenzene yield was estimated as 2.9 mol % . HPLC analyses of the total reaction products from N,Ndimethyl-4-phenylazoaniline(4) and Solvent Yellow 14 (6) indicated the presence of only one peak with a retention time of 2.8 min. Degradation products with HPLC retention times of 2-3 min appeared to be due to watersoluble compounds. Hydrophobic compounds bind strongly to the C-18 HPLC column and, consequently, display longer retention times. However, hydrophilic compounds bind weakly to this column and thus exhibit shorter retention times. For example, aliphatic carboxylic acids have retention times of 2-3 minunder these analytical conditions. Preliminary HPLC and GC/MS analyses of the water-soluble fraction of N,N-dimethyl-4-phenylazoaniline (4) indicated the presence of maleic and formic acids (data not shown).
Table 1. Fea+-HzOz-Catalyzed Degradation of Azo Dyes to COXand Water-Soluble Products* W02 water-soluble compds CH2Cl2-soluble compds (%) (%) (%I 14C substrate
Disperse Yellow 3 OH
(5)
(%)
30
42
19
91
18
31
23
72
16
43
36
95
22
38
28
80
30
43
18
91
21
31
17
69
18
37
35
90
8
63
27
98
16
29
17
62
Disperse Yellow 3 02N-@N=N-@H2
mass balance determined
Solvent Yellow 14
Degradation reactions contained 50 r M 1%-labeled azo dye (50 000-71 OOO cpm), 2 mM ferric nitrate, 1mM nitric acid, and 0.1 % Tween-80, a nonionic detergent. Two additions of 75 mM H2O were made; the first addition was made at 0 h, and the second addition was made at 10 h. Total reaction time was 24 h. An asterisk (*) indicates the location of the I4C label in the azo dye. 14COzvalues reported are the mean values for quadruplicate samples. The first standard deviation for these values ranged from 10.1 to *leg%. For the determination of watersoluble and methylene chloride-soluble compounds, reaction mixtures from all four replicate experiments were pooled and processed as described in the Experimental Section. Mass balance was calculated by adding the radioactive counts in W02,water-soluble compounds, and methylene chloride-soluble compounds.
Identification of Benzene. At the end of each dye degradation experiment, mass balance was calculated by adding the radioactive counts in 14C02,the methylene chloride fraction, and the aqueous fraction (Table 1).This value was in the range of 88-98% for radiolabeled dyes la, 2,3,4a, 5, and 6a. However, for dyes lb, 4b, and 6b in which the phenyl rings were 14Clabeled, the mass balance values were only 72%,69 % ,and 62 % ,respectively (Table 1). In these experiments, the gas phase was analyzed only for 14C02.If the gas phase contained other volatile organic components, they probably were not captured by the C02 traps. In a separate 6-h experiment, the evolution of COZ and volatile organics from the degradation of 14C-labeled dyes was determined simultaneously. All the phenyl ring 14C-labeled azo dyes 4-phenylazophenol (lb), N,N-dimethyl-4-phenylazoaniline(4b), and Solvent Yellow 14 (6b) produced radioactive volatile organics, whereas the aniline ring 14C-labeledN,N-dimethyl-4-phenylazoaniline (4a) and the phenolic ring 14C-labeled4-phenylazophenol (la) did not generate any radioactive volatile organic product (Figure 1). The amount of volatile products
formed from lb, 4b, and 6b degradation in the first 6 h was 8 % ,20%,and 26%, respectively. Mass balance for phenyl ring labeled dyes lb, 4b, and 6b determined after 6-h reaction was 94%,86 % ,and 94 % respectively. These results suggest that the lower mass balances observed for these dyes in our earlier experiments were because the volatile organic compounds were not captured. GC/MS analysis of the gas phase of the reaction mixture from NJV-dimethylphenylazoaniline(6) indicated the presence of only a single volatile product, and the mass spectrum of this compound corresponded to that of benzene. Similar G U M S analyses of the volatile products from phenylazophenol(1) and Solvent Yellow 14 (6) degradation also indicated the presence of only benzene. Azo dyes with substituents on the phenyl azo portion did not generate volatile organic products. Hustert and Zepp identified hydroquinone from UV-Ti02 degradation of 4-phenylazophenol; however, benzene was not identified in that investigation (32). Proposed Mechanism for Benzene Production. Scheme 1 presents a probable mechanism for benzene Envlron. Sd.Technol., Vol. 28, No. 7, 1994
1991
90
Scheme 1. Probable Mechanism for Benzene Generation from 'OH Degradation of Azo Dyes with Phenylazo Substitution: R = "2, OH
a
80
70
--t- Color --t-1 4 ~ - ~ 0 1 a t i l e s
60
-14~02
50
40 30 U
20 10
0
0
2
1
3 4 Time (hr)
5
6
Phenyldiazene
R
R
b 70 1
Aromatic Rinn
Degradation50
I
I
\ 0
1
2
3
4
5
6
Tlme (hr)
Flgure 1. Fe3+-H202dependent degradationof (a) phenyl ring-labeled
N,#dimethyl-4-phenylazoaniline(4b) and (b) aniline ring-labeled N,N
"4
dimethyl-4-phenylazoaniline (44. Decolorization of 4 was monitored at 483 nm: (U) Percentage color remaining; (0)percentage I4COz evolved; (A)percentage volatile organics formed.
formation in the 'OH-mediated degradation of azo dyes. According to our proposal, *OHadds to the azo linkagebearing carbon (C-4) of a hydroxy or an amine-substituted ring. The resulting *OHadduct breaks down to produce phenyldiazene and a phenoxy radical. Phenyldiazene is extremely unstable; *OHor molecular oxygen can readily oxidize it by one electron to yield a phenyldiazene radical (33) (Scheme 1). The latter intermediate is also unstable and cleaves homolytically to generate a phenyl radical and molecular nitrogen (33). The phenyl radical might abstract a hydrogen radical from 'OzH, Tween-80, or dye degradation products to produce benzene. The phenyl radical is not likely to be scavenged by molecular oxygen since it reacts sluggishly with oxygen (34). As for the fate of the phenoxy radical, it might further react with 'OH and oxygen,leading to the aromatic ring degradation. The mechanism proposed for benzene formation could also explain nitrobenzene and acetanilide production from Disperse Orange 3 and Disperse Yellow 3, respectively. Surprisingly, azo dye degradation by 'OH resembles peroxidase-catalyzed degradation of azo dyes described recently by this laboratory (35). 1992 Envlron. Sci. Technol., Vol. 28, No. 7, 1994
Summary and Conclusions This study demonstrates that Fe3+-Hz02 treatment degrades asubstantial portion of azo dyes to water-soluble products and COZ. There is definitive evidence for the generation of benzene when azo dyes with a phenylazo substitution are degraded with FeS+-HzOz. Benzene is a priority pollutant; it causes leukemia in humans and reproductive impairment in fish (36). Several watersoluble commercial azo dyessuch as Naphthol Blue Black, Azogeranine, Chromotrope 2R, and Direct Orange 2G have a phenylazo substitution as part of the dye structure and are likely to generate benzene when degraded with 'OH. Textile industry effluent usually contains a mixture of dyes. This study cautions that, in such a situation, the structure and concentration of individual azo dyes should be known before the effluent is treated using a process which relies on the hydroxyl radical chemistry. Acknowledgments
This research was supported by Grant R819458-01 from the Office of Exploratory Research, U.S. Environmental
Protection AGency, and by the Exxon Education Foundation. We thank Prof. James F. Pankow for providing access to the Finnigan GC/MS facility and Dr. Paul Tratnyek for reviewingthe manuscript. Notice: Although this research is funded in part by the United States Environmental Protection Agency, it has not been subjected to the Agency’s peer and administrative review and, therefore, may not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Literature Cited
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(17)Barbeni, M.; Minero, C.; Pelizetti, E.; Borgarello, E.; Serpone, N. Chemosphere 1987,16,2225-2237. (18)Pignatello, J. J. Environ. Sci. Technol. 1992,26,944-951. (19)Ho,P. C. Enuiron. Sci. Technol. 1986,20,260-267. (20)Lipczynska-Kochany, E. Chemosphere 1992,24,1369-1380. (21)Leung, S.W.; Watts, R. J.; Miller, G. C. J. Environ. Qual. 1992,21,377-381. (22) Murphy, A. P.; Boegli, W. J.; Price, M. K.; Moody, C. D. Enuiron. Sci. Technol. 1989,23,166-169. (23) Walling, C. Acc. Chem. Res. 1975,8, 125-131. (24)Al-Ekabi, H.; Serpone, N. J. Phys. Chem. 1988,92,57265731. (25) Froelich, E. M. In Chemical Oxidations, Technologies for the Nineties; Eckenfelder, W. W . , Bowers, A. R., Roth, J. A., Eds.; Technomic: Lancaster, PA, 1992;pp 104-113. (26) Peyton, G. R.; Glaze, W. H. Environ. Sci. Technol. 1988,22, 761-767. (27) Kuo, W. G. Water Res. 1992,26,881-886. (28) Bigda, R. J.; Elizardo, K. P. Abstracts of Papers, 204th ACS National Meeting, Washington, DC; American Chemistry Society: Washington, DC, 1992;pp 259-262. (29) Moore, S. B.; Antenucci, A. Abstracts of Papers, ACS National Meeting, Denver, CO; American Chemical Society: Washington, DC, 1993;pp 465-466. (30)Environ. Health Perspect. 1993,101 (Suppl. 1))121-123. (31)Spadaro, J. T.; Gold, M. H.; Renganathan, V. Appl. Environ. Microbiol. 1992,58, 2397-2401. (32) Hustert, K.; Zepp, R. G. Chemosphere 1992,24,335-342. (33) Huang, P.-K. C.: Kosower, E. M. J.Am. Chem. SOC.1968, 90,2367-2376. (34)Russell, G. A.; Bridger, R. F. J . Am. Chem. SOC.1963,85, 3765-3766. (35)Spadaro, J. T.;Renganathan, V. Arch. Biochem. Biophys., in press. (36) Sittig, M. Priority Toxic Pollutants-Health Impacts and AllowableLimits; Noyes DataCorp.: Park Ridge, NJ, 1980; pp 87-90. Received for review January 21, 1994.Revised manuscript received April 5, 1994.Accepted April 11, 1994.
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