Environ. Sci. Technol. 1988, 22, 1215-1219
Appel, B. R.; Haik, M.; Kothny, E. L. Atmos. Environ. 1984, 18, 409-416. Atkinson, R.; Aschmann, S. M.; Carter, W. P. L.; Pitts, J. N., Jr. Int. J. Chem. Kinet. 1983,15,721-731. Cadle, R. D.; Schadt, C. J. Am. Chem. SOC.1952, 74, 6002-6004. Japar, S. M.; Wu, C. H.; Niki, H. J.Phys. Chem. 1974, 78, 2318-2320. Adeniji, S. A.; Kerr, J. A.; Williams, M. R. Int. J. Chem. Kinet. 1981, 13, 209-217. Husar, R. B.; Whitby, K. T. Environ. Sci. Technol. 1973, 7, 241-247. Wu, C. H.; Japar, S. M.; Niki, H. J. Enuiron. Sci. Health, Part A 1976, A l l , 191-200. Ohta, T. J. Phys. Chem. 1983,87, 1209-1213.
(27) Darnall, K. R.; Winer, A. M.; Lloyd, A. C.; Pitts, J. N., Jr. Chem. Phys. Lett. 1976,44,415-418. (28) Sakamaki, F.; Akimoto, H.; Okuda, M. Environ. Sci. Technol. 1981,15,665-671. (29) Carter, W. P. L.; Atkinson, R.; Winer, A. M.; Pitts, J. N., Jr. Int. J. Chem. Kinet. 1982, 14, 1071-1103. (30) Grosjean, D. Sci. Total Environ. 1984, 37, 195-211. (31) Friedlander, S. K. In Smoke, Dust and HazeFundamentalsof Aerosol Behavior;Wiley: New York, 1977; pp 243-246. (32) Atkinson, R. Chem. Rev. 1985, 85, 69-201.
Received for review March 26,1987. Revised manuscript received March 1, 1988. Accepted March 22, 1988.
Sequential Degradation of Chlorophenols by Photolytic and Microbial Treatment Raina M. Miller,t George M. Singer,$ Joseph D. Rosen,b and Richard Bartha*vt Department of Biochemistry and Microbiology and Department of Food Science, Cook College, Rutgers University, New Brunswick, New Jersey 08903
w Using the radiolabeled model pollutants 2,4-dichlorophenol (DCP) and 2,4,5-trichlorophenol (TCP) we demonstrated that brief UV (300-nm) photolysis greatly facilitates the removal of the two chlorophenols from sewage through accelerated mineralization and binding of polar products. The addition of 0.1 M HzOzstrongly accelerated the photolysis process resulting in half-lives of 1.68 and 0.87 min for DCP and TCP, respectively. In natural sunlight, half-lives of the chlorophenols were less than 1 day when H20zwas present. During 4 days of incubation in activated sewage sludge, only 3% of unphotolyzed DCP and 1% of unphotolyzed TCP were mineralized. Mineralization rose to 79 and 59%, respectively, after photolysis in the presence of Hz02 Photolysis without H202resulted in removal of chlorophenols from solution chiefly by binding. Increased mineralization and binding were observed also upon incubation of photolyzed chlorophenols in soil. Disruption of carbon-halogen bonds by brief photolysis followed by traditional biological effluent treatment offers an alternative to activated charcoal treatment for removal of xenobiotics from industrial effluents. Introduction
Halo-substituted aromatics occur rarely in natural products but have gained notoriety as environmental pollutants that are resistant to biodegradation (1). Consequently, conventional biological waste treatment is ineffective, and removal from industrial effluents by activated carbon absorption is commonly used (6). Frequent need for carbon reactivation renders this process both inconvenient and costly. The carbon-halogen bonds that prevent or delay biodegradation are, however, susceptible to photolytic cleavage. Unfortunately, total removal of halo aromatics by photolysis is also costly. Economic and mode of action considerations suggested that partial photolysis might prime recalcitrant halo aromatics for subsequent biodegradation. A sequential treatment pro-
'*Department Department of Biochemistry and Microbiology. of Food Science. 0013-936X/88/0922-1215$01.50/0
cess using a relatively brief photolysis period followed by conventional biological waste water treatment may offer both technical and economic advantages. To explore this concept, 2,4-dichlorophenol (DCP) and 2,4,5-trichlorophenol (TCP) were photolyzed with and without added Hz02as a source of hydroxyl radicals. Biodegradation of intact and photolyzed compounds to 14C02and to polar and to bound metabolites was compared. Experimental Section
Photolysis. Uniformly 14C-ring-labeledDCP (sp act. 2.9 mCi/mmol) and TCP (sp act. 9.0 mCi/mmol) were obtained from Pathfinder Laboratories, St. Louis, MO. Their radiochemical purity was determined by thin-layer chromatography using a solvent sytem of hexane:acetone (4:l) on silica gel plates and was found to be 98% for both DCP and TCP. Aqueous solutions (100 pg/mL, w/v) of DCP (1.49 X lo6 DPM/mL) and of TCP (6.32 X lo4 DPM/mL) were irradiated in a Rayonet "merry-go-round" photochemical reactor (Southern New England Ultraviolet Co., Hamden, CT) by using RPR 3000-A lamps. These lamps emit light between 270 and 350 nm with a maximum at 300 nm. There are also small contributions at 248-258, 360-370, 400-410, 430-440, 520-530, and 572-582 nm. Incident intensity, as measued by potassium ferrioxalate actinometry (4), was 4.63 pE m-2 s-l. Prior to irradiation of 14C-labeledmaterial, studies were conducted with aqueous solutions (100 pg/mL) of unlabeled chlorophenols using either the photoreactor or sunlight. These irradiations were conducted in the presence of 0-0.1 M H202. At selected time intervals, 2-pL aliquots of the irradiated chlorophenol solutions were assayed on a Tracor 550 gas chromatograph equipped with FID and a 30 m X 0.53 mm DB-17 vitreous silica megabore capillary column (J & W Scientific, Folsom, CA). A cold on-column injector was used. The oven temperature was kept at 70 "C for 2 min, reset to 110 OC for 2 min, and subsequently programmed to 250 "C at 5 "C/min. Helium carrier gas flow was 12 mL/min. Quantitation was by the external standard method. H202(50%),17.54 M by iodometric titration (B), was added to some of the chlorophenol solutions to give
0 1988 American Chemical Society
Environ. Sci. Technol., Vol. 22, No. 10, 1988 1215
Table I. Fate of Radiocarbon from 2,4-Dichlor~phenol~ during Incubation in Sewageb day 0 1 4 4e
total volatiles UN PH PP 10.0 4.9 6.1 3.4
1.0 8.7
8.5 58.5 81.3 19.3
UN 80.9 85.0 67.6 62.2
nonpolar PH 19.0 11.1
PP
UN
1.0 2.8 2.8 2.4
0.9 2.1 6.9 1.7
polar PH 42.5 38.9
PP
UN
87.7 16.7 14.0 53.5
0.3 0.3 1.6 4.7
bound PH 36.2 45.6
recovery PH
PP
UN
7.9 7.8 12.2 24.5
92.1 92.3 82.2 72.2
PP
97.7
105.1 85.8 116.3 99.7
89.6
OPretreatment symbols: UN = unphotolyzed, PH = photolyzed (30 min), P P = photolyzed (30 min) with HzOz(0.1 M). bReported as percent of added radiocarbon. Poisoned by HgC12.
Table 11. Fate of Radiocarbon from 2,4,6-Trichloropheno1"during Incubation in Sewageb day
0 1 4 4c
total volatiles UN PH PP
UN
3.4 4.6 4.7 3.4
91.6 82.0 81.0 77.3
1.0 7.5
2.2 43.0 62.4 21.3
nonpolar PH 27.9 9.8
PP
UN
1.2 1.4 0.5 0.2
0.4 2.5 3.8 7.1
polar PH 42.5 41.0
PP
UN
57.6 16.3 16.0 57.6
4.0 4.5 3.8 2.4
bound PH 36.3 36.4
recovery PH
PP
UN
1.3 1.7 5.0 1.3
96.7 90.8 94.5 89.1
PP
106.7
65.0 65.2 82.7 69.8
94.7
"Pretreatment symbols: UN = unphotolyzed, PH = photolyzed (30 min), P P = photolyzed (30 min) with HzOz (0.1 M). bReported as percent of added radiocarbon. Poisoned by HgClP
initial concentrations of 0.1 or 0.01 M H202. GC/MS analyses of the photoproducts, obtained in the absence of H202,were performed on a Finnigan MAT 8230 mass spectrometer interfaced to a Varian 3400 gas chromotograph and an SS-300 Finnigan MAT data system. A 30 m X 0.25 mm id., 0.25 pm film thickness DB-5 capillary column was used with on-column injection and programmed between 50 and 250 OC at 4 OC/min. The mass spectrometer was scanned at 1 s/decade between 45 and 450 amu in the E1 (70 eV) mode. The source temperature was 250 "C, and the filament current was 1 mA. Biodegradation. Experiments with intact or photolyzed chlorophenols were conducted in either a sewage sludge or a soil test system. Fresh activated sewage sludge was obtained from the Raritan Valley Sewerage Authority Treatment Plant (Bridgewater, NJ). The floc was allowed to settle. Modified 125-mL microfernbach flasks were charged with intact or photolyzed chlorophenols (3.14 X lo4 to 2.85 X lo5 DPM, corresponding to 0.16-0.24 mg of chlorophenol in various experiments) in 1.8 mL of distilled water. In addition, 1mL of settled sewage sludge floc, 6.2 mL of sewage supernatant, and 1 mL of 0.1 M, pH 7.0, phosphate buffer was added for a total volume of 10 mL in each flask. Poisoned controls received, in addition, 0.2 g of HgC12. All flasks were closed and incubated with gyrotory shaking (150 rpm) at 20 "C. The flasks were flushed with air through a series of six traps at 1-4 days. Traps 1 and 4 were empty in case of backflow, traps 2 and 3 contained a toluene-based scintillation cocktail to trap and scrub out 14C-volatiles,and traps 5 and 6 contained a phenethylamine-based cocktail to trap 14C02(5). To maintain high microbial activity, 0.5 mL of 10-fold concentrated Difco nutrient broth was added to each flask after flushing at 2 days. At 0, 1,and 4 days, flasks were analyzed for distribution of radiocarbon. Each analysis was performed on duplicate flasks as follows: 25 mL of CH2C12was added to each flask and shaken overnight. The flask contents were filtered, and the filter was rinsed with an additional 10 mL of CH2C12. The filtrate was collected in a separatory funnel, and the solvent phase was separated. The aqueous layer was extracted with two more 20-mL aliquots of CH2C1,, and the solvent extracts were combined. The solvent extract was concentrated to 2 mL in a rotary evaporator with a dry ice cooled trap. Aliquots of the trap fluid (volatiles),of the solvent extract (nonpolar materials), and of the aqueous solution (polar 1216
Environ. Sci. Technol., Vol. 22, No. 10, 1988
Table 111. Fate of Radiocarbon from Intact and Photolyzed (30 min) Chlorophenolsa during Incubation in Soilb total
solvent extr bound recovery day UN PH UN PH UN PH UN PH UN PH l4COZ volatiles -
0 21 42 0 21 42
15 18
12 16
22 27
1 17 19
1 26 31
DCP 77 16 13
9 1 1
27 66 64
94 72 66
10 13
1 14 19
1 13 16
TCP 80 12 7 14 6 7
17 51 52
84 58 55
105 104 99 99 96 98 98 72 77
97 85 78
a Pretreatment symbols: UN = unphotolyzed, PH = photolyzed (30 m i d .
Table IV. Effect of Irradiationa Time on the Release of I4CO2and Total Volatiles from Chlorophenols during a 4-Day Incubation Period in Sewageb photolysis time, min
14COz
total volatiles
5 14 18 78
6 15 20 81
1 7 18 51
1 8
DCP 0
5 15 30 TCP 0 5 15 30
18 65
In the presence of 0.1 M HzOz. Reported as percent of radiocarbon added.
materials) were counted. The filtered solids were incinerated by a Model B306 Packard Tri-Carb sample oxidizer, and the evolved 14C02was trapped and counted (bound material). In Tables I and 11, under total volatiles, 14C02 and organic volatiles evolved during incubation and the contents of the dry ice trap from the concentration process are listed together. Net 14C02evolution (mineralization ) is shown in Figure 1 and in Tables I11 and IV. For incubation in soil, fresh Nixon sandy loam ( 3 )was collected and sieved through 2 mm diameter openings without complete air-drying. Of this soil, an amount
Table V. Rate Constants and Half-Lives for Chlorophenol P hotodegradations
80 -
H,O,, M
k, m i d
T ~ , ~ ,
ro
DCP
0.0
TCP
0.01 0.1 0.0 0.01 0.1
0.072 0.177 0.412 0.283 0.321 0.801
9.63 3.92 1.68 2.44 2.16 0.87
0.998 0.997 0.993 0.999 0.982 0.996
compd h
z
DC P
min
Correlation coefficient.
5 1 -
1
2
0
0
3
4
/-
Dol I/ 0
c, J 1
i
were found in emulsions form HgC12-containingflasks. Radioactivity was counted with a p Track Model 6895 liquid scintillation counter (TM Analytic, Elk Grove Village, IL). Oxosol C14 and Betafluor (National Diagnostics, Somerville, NJ) counting fluids were used in trapping 14C02and other volatiles, respectively. All other aqueous and solvent fractions were counted in Scintiverse E (Fisher Scientific, Springfield, NJ). All counts were corrected for background and quenching according to the external standard ratio method.
TCP
Results and Discussion
+
2
3
4
TIME ( D A Y S )
Flgure 1. Mineralization of 2,4-dichlorophenol (DCP) and 2,4,5-trichlorophenol (TCP) during Incubation in the activated sludge test system. Symbols: (0) unphotolyzed chlorophenols, (0) photolyzed chlorophenols (30 min), (H) chlorophenols photolyzed in presence of 0.1 M H,Op (30 mln).
corresponding to 10 g of oven-dry (105 "C) weight was added to modified microfernbach flasks. Aqueous chlorophenol solution or photolysate was added in combination with distilled water to bring the moisture content of the soil to 60% of its water holding capacity (7). The total contents in each flask was 10 g of soil (dry weight) and 3.7 mL of H20. Incubation was at 20 "C for a total of 42 days. The flasks were flushed to collect 14C02and other volatiles twice weekly, and duplicate flasks were analyzed at 0,21, and 42 days. Analysis of soil incubations was similar to that described for sewage incubations. To each flask, 50 mL of CH2C12 and 20 g of anhydrous NaS04 were added and shaken overnight. The samples were filtered, and the filter was rinsed with 25 mL of CH2C12. The combined CH2C12extracts comprised the solvent-extractable fraction. The extracted soil was then incinerated to determine bound 14C02. The CH2C12extracts were concentrated on the rotary evaporator to 2 mL each, and the contents of the dry ice trap were counted and added to the volatiles trapped during the incubation process to yield total volatiles. Extraction efficiency of DCP and TCP from distilled H 2 0 was determined by using three 20-mL aliquots of CH2C12. Further, extraction efficiency of intact and photolyzed DCP and TCP from sewage sludge and soil test systems was determined on day 0 following the entire described analytical procedure for radiocarbon distribution. Results are shown in Tables I, 11,and 111,under nonpolar. Separation of the aqueous and CH2C12phases was clean in most cases, the exception being when HgC1, was present in the incubation flask. In any cases where emulsion occurred, radioactivity in the emulsion was determined separately. No significant (
