Environ. Sci. Technol. 7904, 18, 434-439
Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31. 347-364.
Tokiwa, H.; Nakagawa, R.; Ohnishi, Y. Mutat. Res. 1981, 91, 321-325. Graham, S. C.; Homer, J. B.; Rosenfeld, J. L. J. Proc. R. SOC.London, Ser. A 1975, 344,259-285. "Handbook of Chemistry and Physics", 47th ed.; Chemical Rubber Co.: Cleveland, OH, 1967. Schuetzle, D.; Riley, T. C.; Prater, T. J.; Harvey, T. M.; Hunt, D. F. Anal. Chem. 1981,54, 265-271. Schuetzle, D. paper presented at the EPA Symposium on the Application of Short-Term Bioassays in the Analysis of Complex Environmental Mixtures, Chapel Hill, NC, Jan 25-27, 1982. Wagner, H. G. G. Symp. (Znt.) Combust., [Proc.] 1979,16. Longwell, J. P. Symp. (Znt.) Combust., [Proc.] 1977, 16. Pasternak, M.; Zinn, B. T.; Browner, R. F. Symp. (Znt.) Combust., [Proc.] 1981, 18.
(17) Bittner,J. D.; Howard, J. B. Symp. (Int.)Combust., [Proc.]
1981, 18. (18) Henderson, T. R.; Sun, J. D.; Brooks, A. L. and Bechtold, W. E. paper presented at the 31st Annual Conference on Mass Spectrometry and Applied Topics, Boston, MA, May 5-13, 1983; p 404. (19) Hunt, D. F.; Shabonowitz, J.; Harvey, T. M.; Coates, M. paper presented at the 30th Annual Conference on Mass Spectrometry and Allied Topics, Honolulu, HI, June 1982; pp 800-801. (20) Seizinger, D. E.; Naman, T. M.; Marshall, W. F.; Clark, C. R.; McClellan, R. 0. paper presented at the Society for Automotive Engineers Meeting, Troy, MI, June 1982;paper no. 820813.
Received for review May 24,1983. Accepted November 10,1983. Research performed under US.Department of Energy Contract DE -AC04- 76EV01013.
Fate and Metabolism of Isopropylphenyl Diphenyl Phosphate in Freshwater Sediments Michael A. Heltkamp," James N. Hucklns, Jimmie D. Petty, and James L. Johnson U.S. Department of the Interior, Fish and Wildlife Service, Columbia National Fisheries Research Laboratory, Columbia, Missouri 65201
w The aerobic and anaerobic biodegradation of isopropylphenyl diphenyl phosphate (IPDP) was determined in freshwater sediments with both di[14C]phenyl- and is~propyl[~~C]phenyl-labeled IPDP. Mineralization of IPDP was slow in these sediments, as only about 8% was degraded to I4CO2after 4 weeks. The degradation rates were not affected by oxygen tension, chemical concentration, or seasonal differences in sediment. Chemical analyses of aerobic samples by gas chromatography and mass spectrometry resulted in the tentative identification of nine minor degradation products which suggested pathways involving stepwise demethylation of the isopropyl moiety and enzymatic hydrolysis of both substituted and unsubstituted phenyl moieties, as well as methylation of some intermediates. The diverse microflora occurring in these freshwater sediments appeared to be responsible for the rate and pathway of IPDP degradation. This study indicates that the half-life of IPDP in some freshwater sediments may be greater than previously expected.
Introduction The use of triaryl phosphates (TAPS)in fire-resistant hydraulic fluids and as fire retardant plasticizers has increased steadily over the last 30 years, and the 1977 annual production is believed to have approached 104 million pounds ( I ) . Until the mid-l960s, tricresyl phosphate and cresyl diphenyl phosphate were the predominantly used TAPs. However, these TAPs containing o-cresyl have been associated with neurotoxic effects in mammals (2-4) and poisoning in humans (5-7); furthermore, they are dependent for production on the availability of 0-cresol, and production costs are relatively high. Consequently, isopropylphenyl diphenyl phosphate (IPDP) was introduced as a substitute for the cresyl TAPs. Its production has steadily increased since 1970 ( I ) while that of the cresyl TAPShas decreased. If this trend continues, the annual *Address correspondence to this author at the National Center for Toxicological Research, Jefferson, AR 71602. 434
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Not
production of IPDP is expected to total 60-65 million pounds by 1986 ( I ) . It has been estimated that 70% of the annual production of TAPS is eventually discharged into the environment ( I ) , and most of this industrial effluent goes directly into freshwater ecosystems. Environmental residues of TAPShave been reported in fish tissues (8),water and sediment (9, IO), and drinking water (11). In view of the increased industrial use of IPDP, there is some concern about its environmental fate. The toxicity of IPDP has been determined for freshwater invertebrates (12) and fishes ( I 3 ) ,and although the compound is not acutely toxic a t reported environmental concentrations (9, IO), it is relatively lipophilic (14) and could bioaccumulate through the food chain. The purpose of this study was to determine the rate and probable pathway of IPDP biodegradation in freshwater sediments.
Experimental Section Radiolabeled Compounds. We purchased di[14C]phenyl- and isopropyl[14C]phenyl-labeled2-IPDP from Pathfinder Laboratories Inc., St. Louis, MO. Both lots of IPDP were uniformly ring labeled with a specific activity of 28.97 pCi/mg, and thin-layer and gas chromatography indicated that purity exceeded 99%. The I4C-ring-labeleddiphenyl phosphate (DP) standard was the generous gift of Dr. Derek Muir, Freshwater Institute, Winnipeg, Manitoba, Canada. It had a specific activity of 5.1 pCi/mg, and it was purified by reverse-phase thin-layer chromatography. The purity was verified by gas chromatography after methylation with diazomethane. Low-Exposure Microcosms. Microcosms consisted of 250-mL Erlenmeyer flasks containing 10 g (moist weight) of sediment and 90 mL of lake water (pH 7.1). Sediment was collected with an Ekman dredge from the littoral zone of Little Dixie Reservior, Callaway County, MO. This reservior is slightly eutrophic, well characterized (Table I), and representative of reservoirs in the tilled-plain areas of the Midwest (15,16). The upper 3 cm of sediment was removed, homogenized at low speed in a Waring blender, overlaid with lake water, and stored aerobically in the dark for several days at 22 "C. Each low-exposure microcosm
subject to U.S. Copyright. Published
1984 by the
American Chemical Society
Table I. Microbiological and Chemical Characteristics of Sediment and Water from Little Dixie Reservoir, MO
characteristic total heterotrophs starch hydrolyzers gelatin hydrolyzers nitrate reducers ammonifers
direct bacterial count PH alkalinity total hardness
dissolved organic carbon total nitrogen total phosphorus Ca, Mg, Na K, SO4, C1 a Includes data from the txesent studv.
no. no. no. no.
per per per per no. per no. per
type
sample
value
reference
g g g g g
sediment sediment
mL
water water
4.9 x 104 1.3 X lo3 1.7 x 104 7.8 x 103 2.3 x 103 1.5 x 107 7.1-7.7" 55-65 58-70 5.5 678 54 19, 3, 4 4, 9, 4
15 15 15 15 15 15 15 15, 16 15, 16 15 15 15 16 16
units
mg/L as CaC03 mg/L as CaC03 PdL
PdL mg/L mg/L
was treated with a single application of an environmentally realistic concentration of di[14C]phenyl- or isopropyl[14C]phenyl-labeledIPDP (1.56 pg) in 0.05 mL of acetone. All experiments were conducted with five replicates for each experimental variable (oxygentension, label position), and incubation was for 4 weeks at 22 f 1"C. Microcosms were sterilized by autoclaving and included in the study as controls to enable us to detect abiotic chemical degradation. The rate of mineralization (complete biodegradation) of IPDP was determined by measuring the evolution of 14C02with a flow-through respirometer apparatus (17). To maintain aerobic or anaerobic microcosms, we continuously purged the system with air or nitrogen. At weekly intervals, duplicate 1-mL aliquots were removed from the 50mL monoethanolamine-ethylene glycol (3:7 v/v) solution used to trap 14C02and were added t o scintillation vials containing 15 mL of Fluoralloy-methanol (1:l v/v) scintillation cocktail (Beckman Instrument Co., Fullerton, CA). Radioactivity was measured with a Beckman Model LS230 liquid scintillation counter, and all values were corrected for trapping efficiency, quench, and background. High-Exposure Microcosms. We included high-exposure microcosms in the study to enable the detection of small quantities of IPDP metabolites. These microcosms were prepared, incubated, and sampled as previously described but were spiked with 58.5 pg of di[14C]phenyllabeled IPDP. The rate of 14C02evolution from these high-exposure microcosms was determined by a simplified method modified from Gledhill(18). Glass vials containing 9 mL of 0.2 N KOH were suspended within each closed microcosm, and aerobiosis was maintained by semiweekly swirling and venting. Duplicate 1-mL aliquots were removed from the 14C02trapping solution for sampling and added to scintillation vials containing 15 mL of Fluoralloy scintillation cocktail containing 1mL of Triton X (Beckman Instrument Co., Fullerton, CA), and the radioactivity was measured as described earlier. After the sampling, we discarded the used vials and replaced them with vials containing fresh 14C02trapping solution. The effluents of three replicate microcosms were directed through foam and Tenax columns before 14C02trapping. Analysis of both Tenax and foam plugs revealed no radioactivity resulting from volatilization of undegraded or partly degraded IPDP. Extraction and Analysis. The supernatants from low-exposure microcosms were removed and extracted with three 25-mL volumes of methylene chloride (all solvents were Nanograde and came from Burdick & Jackson Lab-
sediment sediment sediment water water water water water water water
oratories Inc., Muskegon, MI). The combined extracts were added to a 25-mL methylene chloride wash of the glass microcosm flask and dried with anhydrous Na2S0,. Sediments (10 g) were mixed with 100 g of anhydrous Na2S04and placed in a 2-cm i.d. glass column with a glass wool plug and Teflon stopcock. The mixtures were then extracted with 150 mL of methylene chloride at 10 mL/ min, and the eluates were combined with the methylene chloride extracts of sample supernatants. Aerobic sediments were extracted a second time with 150 mL of methanol a t 10 mL/min. All extracts were evaporated at 22 "C to 10 mL, transferred to calibrated test tubes, and evaporated to 5 mL under a gentle stream of dry N2. The extracts were purified with carbon-foam adsorbent columns as previously described (19). Columns of carbon-foam (1 cm i.d. X 10 cm) were prerinsed with consecutive 100-mLvolumes of petroleum ether, toluene, ethyl acetate, and methylene chloride. The methylene chloride sample extracts were added to the column and eluted a t 10 mL/min with 100 mL of ethyl acetate, 100 mL of methanol-toluene (4:l v/v), and 100 mL of acidic methanol (0.1 N HCl in methanol). The carbon-foam extracts were evaporated at 22 "C to 10 mL, transferred to calibrated test tubes, and evaporated to 0.75 mL under a gentle stream of dry N2. Extracts were spotted onto Brinkmann silica gel F-254 precoated thinlayer chromatography plates (20 cm X 20 cm, 0.25 mm thickness, EM Laboratories Inc., Elmsford, NY) and separated by developing twice in hexane-acetone (4;l v/v). Medical no-screen X-ray fiim (Kodak NS-2T) was exposed to thin-layer plates for 7 days and developed to produce autoradiograms. Standard, parent compound, and degradation products from the low-exposuremicrocosms were quantitated by scraping the appropriate band off thin-layer plates, adding the collected particulate to a scintillation vial, and counting as described earlier for the 0.2 N KOH 14C02trapping solutions. Unextracted residues were quantitated by combustion of subsamples of the air-dried, extracted sediment in a biological material oxidizer (R. J. Harvey Instrument Corp., Hillsdale, NJ). Overall efficiency (90.8%) was calculated by adding di[14C]phenyllabeled IPDP at each step of the procedure and determining the final recovery of the spikes off the thin-layer plate. The supernatant and sediment from high-exposure aerobic microcosms were extracted with methylene chloride as described earlier for low-exposure aerobic microcosms. We counted supernatant extracts directly for radioactivity, concentrated the methylene chloride sediment extracts to Environ. Sci. Technol., Vol. 18, No. 6, 1984
435
Table 11. Mineralization' of Isopropylphenyl Diphenyl Phosphate in F r e s h w a t e r Sediments IPDP exposureb
oxygen tension
low low low low high
aerobic aerobic anaerobic anaerobic aerobic
14C-label positionc
1
weeks of incubation 2 3
DP
0.8 (0.2)
2.2 (0.3)
IP
0.0
0.0
DP IP DP
1.4 (0.7)
3.1 (1.0)
0.0 0.7 (0.2)
0.0 3.3 (0.7)
5.0 0.5 5.1 0.6 6.0
4
(1.1)
7.1 (0.9)
(0.1)
2.0 (0.2) 7.3 (2.4) 1.1(0.7) 8.4 (3.1)
(1.6)
(0.3) (2.1)
'Values are the mean (one standard error in parentheses), expressed as total percent degraded; however, actual statistical analyses were performed on log transformed data. Concentrations for low and high are 15.6 and 585 fig/L, respectively. D P and IP represent di[14C]phenyl- and is~propyl[~~C]phenyl-labeled IPDP, respectively.
10 mL by rotary evaporation, and purified 5-mL subsamples by gel permeation chromatography (GPC). The GPC column (glass 2.5 cm i.d. X 50 cm) was packed with Biobeads S-X3; the mobile phase was a mixture of methylene chloride and cyclohexane (1:l v/v). Radioactive residues of IPDP eluted a t 5 mL/min in the 100-210-mL GPC fraction. The GPC eluates were concentrated to 5 mL and further purified on columns of carbon-foam as described earlier. Purified samples were then analyzed by gas chromatography (GC) with a flame photometric (FPD) or a thermionic specific detector (TSD). Methanol extracts of the high-exposure sediments (second extract) were concentrated by rotary evaporation to 10 mL and then transferred to calibrated test tubes and evaporated to 1mL under dry N2. Subsamples (500 pL) of the methanol concentrates were chromatographed on a preparative CIShigh-performanceliquid chromatography (HPLC) column (10 pm adsorbosil from Milton Roy Co., Riviera Beach, FL; 2.36 cm i.d. X 20 cm). All separations were performed isocratically a t 2.5 mL/min, and the column was eluted sequentially with 75 mL of 30% water in methanol, 50 mL of methanol, 50 mL of ethyl acetate, 50 mL of methanol, and finally 200 mL of the initial mobile phase. Radiolabeled DP eluted in the 40-45-mL fraction of the methanol-water mobile phase (first column eluate), whereas IPDP eluted in the ethyl acetate mobile phase. Polar 14C-labeledresidues in the methanol-water eluates were concentrated with acetonitrile to remove water and then methylated with diazomethane. These samples were examined on FPD-GC and characterized by a capillary column gas chromatograph-mass spectrometer (GC-MS). Radioactive residues in the ethyl acetate eluates of the preparative C18 column were concentrated by rotary evaporation and analyzed by FPD-GC. Gas Chromatography and Mass Spectrometry. A Varian Model 3700 GC (Varian Instruments, Walnut Creek, CA), equipped with FPD and TSD detectors, was used in the phosphorus mode for analysis of the purified sediment extracts from the high-exposure microcosms. The GC columns were glass, 2 mm i.d. X 1.1m, with 3% OV-101 (TSD) or SE-30 (FPD) on Chromosorb W HP. Analyses were performed isothermally a t 170 "C and programmed from 140 (2 min isothermal) to 250 "C a t 10 "C/min and held a t 250 "C for 5 min. A Finnigan Model 4023 GC-MS system (Finnigan Instruments, Sunnyvale, CA), equipped with a quadrapole mass filter and a DB5 capillary column (J & W Scientific, Rancho Cordova, CA; 0.25 mm i.d. X 30 m), was used to characterize degradation products of IPDP. Analyses were performed in the electron impact mode, and the electron energy was set at 55 eV. Samples were injected at 60 "C, held isothermally for 2 min, and programmed to 210 OC a t 20 "C/min. The program rate was then reduced to 5 "C/min to 290 "C, and the sample was held isothermally a t 290 "C for 15 min. 436
Environ. Scl. Technol., Vol. 18, No. 6 , 1984
Statistics. The data were analyzed as a split plot in time in which the main plot contained the main effects of oxygen tension and label position and the interaction of oxygen and label position. The subplot contained the main effect of time and all possible interactions of oxygen tension, label position, and time. Fisher's Protected Least Significant Difference (LSD) was used to determine differences between means.
Results IPDP Mineralization. Isopropylphenyl diphenyl phosphate (IPDP) degraded slowly in freshwater sediments. After 4 weeks I4CO2evolution from di[14C]phenyland isopropyl[ 14C]phenyl-labeledIPDP was only 7-8% and 1-2%, respectively (Table 11). Examination of these biodegradation rates reveals a steady-state, nearly linear rate of biodegradation for IPDP that probably represents first-order kinetics. Oxygen tension had no significant effect on IPDP degradation. The evolution of 14C02from i~opropyl[~~C]phenyl-labeled IPDP was not detected for the first 2 weeks of the study and lagged 2 4 % behind the 14C02evolution observed for di[14C]phenyl-labeledIPDP during the last 2 weeks. The analysis of variance revealed a highly significant interaction of label position X time, which indicated that the diphenyl and isopropylphenyl moieties of IPDP were not mineralized at the same rate. No radioactivity resulting from abiotic degradation or volatilization of IPDP was observed in sterilized controls. Water and sediment from low-exposure microcosms (Table 111)were analyzed with thin-layer chromatography. Extraction with methylene chloride led to the recovery of 80% of the radioactivity present in anaerobic sediments but only 52-56% of that in aerobic sediments. Subsequent extraction of aerobic sediments with methanol resulted in the recovery of an additional 30% of the radioactivity. However, examination of the methylene chloride extracts and each of the three carbon-foam fractions of the methanol extracts (aerobic sediments) with thin-layer chromatography indicated that nearly all these residues were IPDP. The extracted radioactivity had an R, value (0.68) identical with that of the IPDP standard. Only the ethyl acetate fraction contained a small amount of 14Clabeled residue (1.2%) that had an R value (0.62) slightly less than that of the IPDP standard (0.68). From these analyses, we conclude that most of the radioactivity extracted from these sediments was IPDP. IPDP Metabolism. The residue composition and degradation of IPDP in high-exposure microcosms appeared to parallel that in low-exposuremicrocosms (Table I1 and 111). Polar 14C-labeledresidues, which included radioactivity remaining in microcosm water after methylene chloride partitioning plus the methanol-water fractions of HPLC separations of the methanol extracts of sediment, represented only 2.4-3.970 of the total radioactivity spiked into microcosms. Triphenyl phosphate
Table 111. Distribution of IPDP Residues" in Sediment and Water Microcosms after 4 Weeks of Incubation
IPDP exposureb
oxygen tension
I4C-label positionC
degraded
to 14c02
extracted residues in sediment and water
low low low low low, sterile high
aerobic aerobic anaerobic anaerobic aerobic aerobic
DP
7.1 2.0 7.3 1.1 0.0 8.4
82.2 86.6 79.7 80.8 86.7 76.1
IP DP IP DP DP
nonextracted residues in sediment
total recovered radioactivity
5.8 3.9 2.8 d 9.2 2.7
95.1 92.5 90.0 81.9 95.9 87.2
Concentrations for low and high are 15.6 and 585 pg/L, a Values are the mean expressed as percentage of the total radioactivity. respectively. D P and IP represent di[14C]phenyl- and is~propyl['~C]phenyl-labeledIPDP, respectively. No data available.
Table IV. Tentative Identification of Minor Radiolabeled Metabolites Isolated from Microcosm Sediments 4 Weeks after a Single Application of 585 pg/L IPDP scan no.
GC retention time, min
mass spectrum m / z (% of base peak)
molecular formula
assignment
402 431 440
13.40 14.37 14.67
C13H1304P C14H1504P C14H1504P
diphenyl phosphate (DP) methyl-DP, isomer 1 methyl-DP, isomer 2
456 464
15.20 15.47
Cl6HlgO4P C15H1704P
isopropyl-DP ethyl-DP or dimethyl-DP
495
16.50
Cl6HI9O4P
3-carbon-substituted D P
517
17.23
Cl7HZ1O4P
4-carbon-substituted D P
528
17.60
Cl7HZ1O4P
4-carbon-substituted D P
544
18.10
264 (M+., 38), 170 (38), 91 (35), 77 (loo), 65 (34) 278 (M+-, loo), 184 (28), 165 (480), 105 (22), 77 (49) 278 (M+., 85), 184 (28), 169 (60), 131 (22), 106 (31), 91 (loo), 77 (53) 306 (M+*, 42), 189 (65), 118 (loo), 77 (25) 292 (M+., loo), 179 (30), 165 (39), 105 (39), 96 (26), 91 (30), 83 (28), 77 (33), 69 (41), 55 (62) 306 (M+., loo), 291 (65), 179 (42), 104 (24), 90 (27), 77 (22) 320 (M+:,80), 215 (49), 193 (1001, 179 (481,121 (42), 105 (82). 77 (63) 320 (M+.,'lOO),'217 (22), 193 (451, 179 (21), 118 (241, 105 (30), 91 (23), 77 (28) 320 (M+*,95), 305 (42), 215 (32), 193 (43), 179 (231, 118 (24), 105 (40), 91 (21), 77 (52), 71 (31), 57 (56)
C17H2104P
t-butyl D P
(TPP) was the only degradation product detected in nonpolar fractions of purified samples, and it represented only 3.4-13.2% of the radioactivity applied (Figure 1). Some 14C-labeledresidues were observed in the supernatant of the sterilized control, but only background residues were observed in the methanol-water HPLC fraction. Small amounts of polar residues (
