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Naturally Occurring Proteins from Pond Water Sensitize Hexachlorobenzene Photolysis. Monlka Hlrsch and Otto Hutzinger". Chair of Ecological Chemistry ...
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Environ. Sci. Technol. 1989, 23, 1306-1307

Servos, M. R.; Muir, D. C. G. Environ. Toxicol. Chem. 1989, 8, 141-150. Landrum, P. F.; Nihart, S. R.; Eadie, B. J.; Gardner, W. S. Environ. Sci. Technol. 1984, 18, 187-192. Yin, C.; Hasset, J. P. Environ. Sei. Technol. 1986, 20, 1213-1217. Burkhard, L. P.; Kuehl, D. W. Chemosphere 1986, 15, 163-167. Servos, M. R. Ph.D. Thesis, University of Manitoba, Winnipeg, Manitoba, Canada. Dunnivant, F. M.; Coates, J. T.; Elzerman, A. W. Environ. Sei. Technol. 1988, 22, 448-453. Mackay, D.; Shiu, W. Y.; Sutherland, R. P. Environ. Sei. Technol. 1979, 13, 333-337. Webster, G. R. B.; Friesen, K. J.; Sarna, L. P.; Muir, D. C. G. Chemosphere 1985, 14, 609-622. Podoll, R. T.; Jaber, H. M.; Mill, T. Environ. Sei. Technol. 1986,20,490-492. Friesen, K.; Sarna, L. P.; Webster, G. R. B. Chemosphere 1985,14, 1433-1440. Karickhoff, S. W.; Brown, D. S. J . Environ. Qual. 1978, 7, 246-252. McCarthy, J. F.; Black, M. C. 10th Symposium on Aquatic Toxicology and Hazard Assessment; Adams, W. J., Chapman, J. A., Landis, W. G., Eds.; S.T.P. 971, A.S.T.M.:

Philadelphia, PA, 1987; pp 233-246. (26) Chiou, C. T.; Kile, D. E.; Briton, T. I.; Malcolm, R. L.; Leenheer, J. A.; MacCarthy, P. Environ. Sei. Technol. 1987, 21, 1231-1234. (27) Malcolm, R. L.; MacCarthy, P. Environ. Sei. Technol. 1986, 20,904-911. (28) Roa, P. S. C.; Davidson, J. M. In Environmental Impact of Nonpoint Source Pollution; Overcash, M. R., Davidson, J. M., Eds.; Ann Arbor Science: Ann Arbor, MI, 1980; p 23. (29) Bowman, B. T.; Sans, W. W. J . Environ. Qual. 1985, 14, 265-269. (30) Bowman, B. T.; Sans, W. W. J . Environ. Qual. 1985, 14, 270-273. (31) Karickhoff, S. W. In Contaminants and Sediments; Baker, R. A., Ed.; Ann Arbor Science: Ann Arbor, MI, Vol. 11, pp 193-205. (32) Karickhoff, S. W.; Morris, K. R. Environ. Toxicol. Chem. 1985,4,469-479.

Received for review July 14,1987. Revised manuscript received June 21, 1988. Accepted March 30, 1989. W e are grateful to NSERC and Canada Dept. of Fisheries and Oceans for financial support.

Naturally Occurring Proteins from Pond Water Sensitize Hexachlorobenzene Photolysis Monlka Hlrsch and Otto Hutzinger"

Chair of Ecological Chemistry and Geochemistry, University of Bayreuth, 8580 Bayreuth, FRG Photolysis of hexachlorobenzene (HCB) in aqueous solution was conducted with several naturally occurring substances as sensitizer. The reaction rate was increased especially by aromatic amines such as tryptophan and diphenylamine. A protein mixture extracted from surface water was found to have a high sensitizing effect on the photolysis of HCB. Pentachlorobenzene was the only detectable transformation product. Introduction

Photolysis is one important sink for chlorinated hydrocarbons in the aquatic environment. Hexachlorobenzene (HCB), as a representative of this class of chemicals, is persistent in natural systems (1). The photolytic behavior of HCB in aqueous solutions has not been extensively investigated, presumably due to its low water solubility (-3 pgL-'). The light-induced degradation of HCB in distilled water was investigated by Dime (2) and in a mixture of acetonitrile/water by Choudry and Hutzinger (3). In both cases the main transformation product was pentachlorobenzene. The photolysis of HCB was not sensitized by acetone, as is the case with tetra- and pentachlorobenzene ( 3 ) . To estimate the relevance of photolytic degradation of HCB in the aquatic environment, photolysis experiments with lake water extracts and with several naturally occurring substances as sensitizer were carried out. Experimental Section

Irradiation Equipment. Irradiation was carried out in a Rayonett photochemical reactor with a merry-goround apparatus. The lamps used were low-pressure mercury lamps (RPR 300) with a maximum energy output at X = 290-310 nm. The reactor was equipped with eight Pyrex test tubes to cut off wavelengths below 280 nm. Irradiation intensity was measured by uranyl oxalate ac1306

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tinometer to check for constancy of irradiation conditions. Intensity varied at most by a factor of 2 between experiments. A dark control was monitored for each experiment. No dark reaction was observed in any case. Preparation of Aqueous Solutions of HCB. From a concentrated stock of HCB in hexane an aliquot was placed in an Erlenmeyer flask. The hexane was evaporated under a stream of nitrogen. Distilled water, pond water, or protein solution was then added to give a solution of -3 1g-L-l HCB. The solution was shaken for at least 48 h. The sensitizers (diphenylamine, tryptophan, skatole) were added directly before irradiation as a concentrated aqueous solution. Analysis of HCB. All chemicals used were analytical grade except hexane, which was double distilled from technical grade. Samples were acidified with concentrated HC1 to pH = 1 and extracted by shaking three times with 10 mL of hexane. The extract was dried with anhydrous sodium sulfate and evaporated under a stream of nitrogen. HCB concentrations were quantified by GC/ECD with external standard. Pentachlorobenzenewas identified by GC/MSD and external standard as a reference for retention time. Extraction of Total Protein from Pond Water. Pond water (9 L) from the pond located at the University of Bayreuth (pH = 7.4) was filtered under pressure through cellulose acetate filters (molecular weight cutoff, 10000). Distilled water (300mL) was added to the filtration residue and the solution was shaken. The suspension was allowed to settle for 24 h. Protein precipitaton from the supernatant was conducted by addition of (NH4)$04 (70% solution). The precipitate was separated from the salt solution by centrifugation (4000 rpm, 10 min). The centrifugate was dissolved in -10 mL of buffer, pH = 7.4 (4), and dialyzed four times against 1 L of buffer solution (molecular weight cutoff of dialysis membrane, 1000). Total protein content was determined by Bic-Rad protein


0 1989 American Chemical Society

Table I. First-Order Rate Constants for All Photolysis Experiments


rate constant, s-'

distilled water tryptophan, 3.4 kg.L-' pond water skatole, 3.3 mg.L-' protein, 0.6 mgL-' tryptophan, 3.4 mgL-' diphenylamine, 1 mgL-'

1.3 X 2.3 X 2.9 x 6.7 X 8.1 x 1.7 X 7.2 X


10" 10-5


lo-' lo-'

assay. The absorption of the dye protein complex was measured photometrically at h = 595 nm. Bovine albumin served as reference substance. Results and Discussion

Direct Photolysis of HCB in Distilled Water. Photolysis in distilled water was conducted to obtain a nonsensitized photolysis rate for HCB. This rate was compared with those from experiments with sensitizers. The photolysis first-order rate constant of the reaction was

k = 1.3 X lo4 (s-l) No transformation product could be identified. The rate expressions of all further experiments were treated in first-order form. This is the case with a very low concentration of reactive chemical and a constant high concentration of sensitizer in comparison to it (5). Diphenylamine, Tryptophan, and Skatole. Table I lists the first-order rate constants of all performed experiments. Diphenylamine (1 mgL-'), tryptophan (3.4 mgL-' and 3.4 figL-'), and skatole (3.3 mg.L-9 were examined as sensitizers for HCB in aqueous solution. All three sensitize the photolytic reaction of HCB, with diphenylamine increasing the photolysis rate the most. Plimmer et al. (6) demonstrated the photosensitizing effect of diphenylamine for HCB adsorbed on silica gel with wavelengths h > 260 nm. The mechanism of reaction is explained by formation of a charge-transfer complex, which suggests that the reaction is an amine-induced photolysis. Therefore, naturally occurring substances containing amine groups were investigated as sensitizers for HCB. As a test substance, tryptophan was chosen. Its sensitizing potential was previously demonstrated by Soderquist (7) in the photodegradation of the thiocarbamate herbicide molinate. The photolysis rate constant of HCB increases with increasing tryptophan content, but tryptophan still sensitizes the HCB photolysis in the lower ppb range, which is a concentration occurring in lakes (8). Skatole (3-methylindole)was tested to examine whether the amino group or the indole ring of tryptophan acts as a sensitizer. The concentration of skatole was 3.3 mgL-'. The photolytic first-order rate of the reaction was similar to that of the tryptophan-sensitized reaction. This suggests that the photocatalytic reaction is mainly due to the indole ring. The only transformation product that could be identified in the experiment with skatole was pentachlorobenzene (PCBz). The yield of PCBz produced was 18% as related to the starting concentration of HCB. Protein Mixture Extracted from Surface Water. The experiments with aromatic amines raised the question

of whether proteins also influence the photolytic decay of HCB. Therefore a crude protein mixture extracted from surface water was investigated for its photosensitizing effect on HCB. HCB was irradiated in an aqueous solution of extracted protein (0.6 mgL-') and dissolved in original pond water (pond located at the University of Bayreuth, pH = 7.4, protein content = 80 pgL-'). In both cases the photolysis of HCB is accelerated. The enhanced photolysis rate of the protein-sensitized photolysis over that in pond water suggests that the sensitizing effect of pond water is largely due to dissolved proteins. Again the only transformation product that could be detected was PCBz. The yield of produced PCBz was 9% in pond water and 12% in the solution of extracted protein. No polar compounds, as for example, pentachlorophenol, which are expected to be transformation products of the photolysis of HCB in aqueous solution (9),could be detected. Error ranges for all photolysis first-order rate constants were between 25 and 35% as was determined by double and triple experiments. The following conclusions can be drawn: The photolysis rate of HCB is increased by at least 1 order of magnitude by all nitrogen compounds investigated. Tryptophan functions as sensitizer for HCB even at the low concentrations that exist in natural surface waters. Dissolved proteins may play an important role in sensitizing the photolytic degradation of HCB in aqueous systems. The only transformation product detected, PCBz, does not account for all the loss of HCB in the photolysis experiments. Another possible product could be pentachlorophenol, whose photolytic half-life in aqueous systems is too short (10) to be detectable. Registry No. HCB, 118-74-1; PCBz, 608-93-5; tryptophan, 73-22-3; diphenylamine, 122-39-4; skatole, 83-34-1.

Literature Cited (1) Water-Related Environmental Fate of 129 Priority Pollutants; EPA-440/4-79-029b; U.S. Environmental Protection Agency: Washington, DC, 1979; Vol. 11. (2) Dime, R. A. Dissertation, University of California, Davis, 1982. (3) Choudry, G. G.; Hutzinger, 0. Environ. Sci. Technol. 1984, 18, 235. (4) OECD-Guidelines for Testing of Chemicals, Section 1, Physical-Chemical Properties; OECD: Paris, 1981. (5) Zepp, R. G.; Baughman, G. L.; Schlotzhauer, P. F. Chemospere 1981, 10, 119. (6) Plimmer, J. R.; Klingebiel, U. I. J . Agric. Food Chem. 1976, 24, 721. (7) Soderquist, C. J.; Bowers, J. B.; Crosby, D. G. J . Agric. Food Chem. 1977,25, 940. (8) Hutchinson, G. E. A Treatise on Limnology; John Wiley & Sons, Inc.: New York, 1957. (9) Mill, T.; Haag, W. In Hexachlorobenzene: Proceedings of an International Symposium; Morris, C. R., Cabral, J. R. P., Eds.; IARC Scientific Publications No. 77; International Agency for Research on Cancer: Lyon, France, 1986; p 61. (10) Wong, A. S.; Crosby, D. G. J . Agric. Food Chem. 1981,29, 125. Received for review July 6, 1988. Accepted June 2, 1989.

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