Environ. Sci. Technol. 1985, 19, 282-285
possible application of our dechlorination method are in progress (in cooperation with Professor M. Tels and M. M. G. Senden of Eindhoven Technical University, The Netherlands).
Acknowledgments Arochlor 1248 and compositional information was obtained from H. Compaan (TNO, Central Laboratory, Delft, The Netherlands). Registry No. PhC12,25321-22-6;PhCl,, 12002-48-1;Phz, 9252-4; ClPht, 27323-18-8; PhC1, 108-90-7; 1,2,4-C&Ph,120-82-1; Aroclor 1248, 12672-29-6; benzene, 71-43-2.
Literature Cited (1) Louw, R.; Lucas, H. J. Recl. Trav. Chim. Pays-Bas 1973, 92, 55-71. (2) Louw, R.; Rothuizen, J. W.; Wegman, R. C. C. J . Chem. SOC.,Perkin Trans. 2 1973, 1635-1640. (3) Van Nierop, K.; Kuyers, F. J.; Vonk, W. F. M.; Louw, R. J . Chem. SOC., Perkin Trans. 2 1977,1062-1066.
Dorrepaal, W.; Louw, R. Int. J . Chem. Kinet. 1978, 10, 249-275. Van Nierop, K.; Louw, R. Recl. Trav. Chim. Pays-Bays 1977, 96, 176-178. Vonk, W. F. M. Ph.D. Thesis, Leiden, 1980. Mulder, P.; Louw, R. Tetrahedron. Lett. 1982, 23, 2605-2608. Louw, R.; Dijks, J. H. M.; Mulder, P. Chem. Ind. 1983, 759-760. Louw, R.; Dijks, J. H. M.; Mulder, P. R e d . Trav. Chim. Pays-Bas 1984,103, 271-275. Stull, D. R.; Westrum, E. F.; Sinke, G. C. “The Chemical Thermodynamics of Organic Compounds”;Wiley: New York, 1969. Sauer, M. C., Jr.; Mani, I. J . Phys. Chem. 1970, 74,59-63. Pryor, W. A.; Lin, T. H.; Stanley, J. P.; Henderson, R. W. J. Am. Chem. SOC. 1973, 95, 6993-6998. Louw, R.; Dijks, J. H. M.; Manion, J. A.; Mulder, P., unpublished observation. Received for review April 3, 1984. Accepted October 11, 1984. Financial support was given by The Dutch Ministry of Environmental Affairs (VROM).
a-Tocopheryl Acetate as an Indicator of Municipal Waste Contamlnation in the Environment7 Robert P. Eganhouse” and Isaac R. Kaplan
Department of Earth and Space Sciences and Institute of Geophysics and Planetary Physics, university of California, Los Angeles, California 90024 During studies of the extractable organic matter in municipal wastes of southern California, a-tocopheryl acetate (vitamin E acetate) was found to be present in all of the 27 samples tested. The concentration of a-tocopheryl acetate in effluents receiving mainly primary treatment ranges from 6 to 25 pg/L, whereas sludge samples contain approximately 1100 pg/L. This compound is produced industrially for a variety of commercial purposes and can readily be distinguished from natural atocopherol which occurs only as the free alcohol. Its occurrence in municipal wastes reflects the widespread domestic use and disposal of vitamin E acetate-bearing products. Thus, when observed in the environment, the acetate derivative is an indicator of sewage contamination. An examination of sediments deposited in the vicinity of a submarine waste outfall system clearly revealed the presence of a-tocopheryl acetate, suggesting that this molecule can survive the earliest stages of diagenesis.
Introduction One of the central problems facing organic geochemists is that of reconstructing the depositional and diagenetic histories of recent sediments. In coastal areas adjoining urban centers this task is especially challenging because of the complex nature and large number of inputs involved. To further complicate matters, early alteration of the organic matter occurs in the water column and surface sediments, frequently eliminating indicator compounds. Consequently, the bulk‘compositions of the inputs often appear to be similar. Under these circumstances the ‘Publication No. 2504 of the Institute of Geophysics and Planetary Physics, University of California at Los Angeles. * Address correspondence to this author at the Environmental Sciences Program, University of Massachusetts, Boston, MA 02125. 282 Environ. Sci. Technol., Vol. 19, No. 3, 1985
differentiation of organic inputs to sediments can become an arduous, if not intractable, analytical problem. One potentially powerful means of overcoming these difficulties is through the use of molecular tracers, compounds which undergo degradation slowly and whose chemical structures link them uniquely to a given source material or mode of origin. Because a large fraction of all but the most refractory of organic substances can be rapidly decomposed in marine waters, these two requirements are quite stringent and, in practice, rarely satisfied. In the course of our studies of the extractable organic matter in municipal effluents of southern California, we found a group of synthetic hydrocarbons, the linear alkylbenzenes, which appear to satisfy these criteria (1). We recently presented data of their occurrence in wastes, as well as marine sediments and suspended particulates collected in coastal waters near a submarine municipal waste discharge (2). This report constitutes an extension of that work; however, it concerns another potential sewage tracer, a-tocopheryl acetate, hereafter referred to as a-TA. Our purpose in this writing is to provide early evidence for the presence of a-TA in municipal wastes and in coastal sediments known to be contaminated by sewage. To our knowledge, these data represent the fiist such observations to appear in the literature.
Experimental Section Sampling. Samples of wastewater effluent were obtained in 1979 from the four major municipal treatment plants in southern California. A description of the waste treatment procedures, sampling dates, and the methodology used in collecting effluent is given in an earlier publication (3). Subsequent to the effluent sampling, marine Sediments were recovered by gravity coring in the vicinity of a submarine outfall system associated with one
0013-936X/85/0919-0282$01.50/0
0 1985 American Chemical Society
of the treatment plants. The station location and methods of sampling are described in another paper and, therefore, will not be elaborated here (2). Lipid Extraction, Chromatographic Separations, and Analysis, What follows is a brief account of the procedures used in the analysis of effluent and sediment samples for a-tocopheryl acetate (2H-1-benzopyran-6-01, 3,4-dihydro-2,5,7,&tetramethyl-2-(4,8,12-trimethyltridecyl) acetate). A more detailed presentation can be found elsewhere (4,5). The wastewater effluent samples were adjusted to pH 1 (HC1) and extracted with hexane and CHCIS. The combined extracts were then concentrated by rotary evaporation, dehydrated (Na2S04),treated for sulfur removal (activated CuO), and esterified (BF8MeOH). Separation of the extracted lipids into four fractions was achieved by using silica gel thin-layer chromatography (CH2C12development). The ester fraction containing a-TA (fraction F,) was measured gravimetrically and subsequently analyzed-by high resolution fused silica capillary gas chromatography (HRGC) and HRGC/mass spectrometry (electron impact mode). The chromatographic and mass spectrometric conditions are published separately (I, 4). Tentative assignment of structure to the peak believed to be a-TA was based on computer matching of the mass spectrum of the sample peak with library spectra (NBS/EPA library) as well as that of authentic dl-a-tocopheryl acetate (Fluka Chemical Co.). Figure l a displays the structure of a-tocopheryl acetate and the mass spectrum that was obtained by analysis of the authentic standard. The M+ - 42 ion is characteristic of the acetate moiety. Shown in Figure l b is a representative gas chromatogram of an F2fraction isolated from waste effluent and the mass spectrum corresponding to the peak identified as a-tocopheryl acetate. Additional confirmation was obtained by coinjection of the a-TA standard with Fz fractions from waste and sediment samples on a DB-5 fused silica capillary column (30 m X 0.26 mm i.d., 0.25-pm film thickness; J&W Scientific Co.). Quantitation of a-TA in waste samples by HRGC was achieved by comparing integrated peak areas with an external calibration standard run the same day. No correction was made for recovery. Preliminary Studies. Our initial discovery of a-TA in all F2fractions of 27 waste samples was cause for both surprise and concern. One of our concerns was the possibility that a-TA might have arisen as a procedural artifact. Contamination of the samples was not indicated as a-TA could not be detected in processed blanks. A second possible source we considered was the production of a-TA via acetylation of a-tocopherol. However, this is not indicated by our results and is unlikely for two reasons. First, if a-tocopherol was actually present in waste samples, a fact which is as yet unconfirmed, its reaction with acetic acid would proceed more slowly than for a primary or secondary alcohol. Sterols (which are abundant in waste samples) would therefore be expected to undergo acetylation to form the corresponding steryl acetates as coartifacts. The same logic can be applied to other alcohols. The fact is, however, that a-TA is the only acetate found in these samples. This suggests that the acetylation reaction is not operative. A second line of evidence comes from the fact that a-TA was found in waste samples carried through a simplified procedure consisting essentially of a sequence of physical separations (extraction, dehydration, adsorption chromatography, ¬reatment"; see below). Under the mild conditions employed, it is difficult, if not impossible, to envision a mechanism whereby substantial amounts of only one alcohol could be acetylated.
a
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247
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' 500
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/
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/I
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TIMEFigure 1. (a) Structure of a-tocopheryl acetate and corresponding electron impact (70 eV) mass spectrum for all-racemic a-TA mixture (dl-a-tocopheryl acetate). Stars indlcate asymrnetrlc carbons. (b) Gas chromatogram of the F, fraction isolated from waste effluent wlth electron Impact mass spectrum corresponding to the peak identifled as a-tocopheryl acetate. Normal saturated long-chain fatty acld methyl esters deslgnated as n:O where n = number of carbons in alkyl chain.
We therefore conclude that the a-TA in these samples is a true sample constituent and not an artifact generated by our methodology. We were also surprised that this ester had survived the transesterification step and concerned that this might indicate an incomplete conversion of the glycerides in our waste samples to glycerol plus methyl esters of the fatty acids. Because a-TA is an ester of an aromatic alcohol, one would expect it to have a lower reactivity to transesterification than esters formed from primary alcohols, presumably due to steric hindrance. However, the extent of reactivity, and hence recovery, was unknown. We therefore performed a brief experiment to test the effect of various procedures on the recovery of a-TA from waste samples. Aliquots of wastewater extract were given one of three treatments prior to adsorption chromatography: (1)no treatment, (2) a saponification treatment (5) followed by esterification with BF3-MeOH, and (3) the BF,-MeOH transesterification used in the original procedure (4). The yields achieved by saponification/esterification and esterification treatments were then compared with that obtained by using no treatment. As expected, the saponification/esterification procedure resulted in complete hydrolysis of the ester linkage and consequent Environ. Scl. Technol., Vol. 19, No. 3, 1985
283
Table I. Concentrations of a-Tocopheryl Acetate Measured in Waste Effluent Samples Taken in Southern California during 1979=
waste effluentb JWPCP
sampling date 1/15/79 5/15/79 7/16/79 12/13/79
f f S
Hyperion 5-mile
1/15/79 4/18/79 7/16/79 10/16/79
f f S
Hyperion 7-mile
1/15/79 4/18/79 7/16/79 10/16/79
Z f S
OCSD
1/15/79 4/12/79 7/16/79 10/16/79
f f S
SDSD
1/16/79 4/17/79 7/17/79 10/16/79
f f S
concn of a-tocopheryl acetate rg/g of rg/L so1ids 9.7 55 10 61 13 59 9.1 57 10 f 2 58 f 3 6.0 82 21 220 13 170 25 360 16 f 8 210 f 120 1100 120 1100 160 1100 160 1200 140 1100 i 50 140 f 19 9.1 51 20 170 8.9 63 14 110 13 f 5 98 f 54 13 81 18 170 15 70 18 170 16 f 2 120 f 55
n-C,s:o acidla-TA 280 NDd 320 NDd 300 f 28 310 130 150 93 170 f 96 44 20
per capitae emission rate, mg/(cap-day)
4.0
14
18
19 25 f 12 170 99 260 170 170 f 66 170 190 200 200 190 f 14
5.4
7.5
“Also given are stearic acidla-tocopheryl acetate ratios and mean per capita mass emission rates of a-tocopheryl acetate. * JWPCP (Joint Water Pollution Control Plant, County of Los Angeles), Hyperion 5-mile, Hyperion 7-mile (City of Los Angeles), OCSD (Orange County Sanitation District), and SDSD (San Diego Sanitation District). See ref 3 for more details of effluent characteristics. Calculated from measured values of the total suspended solids assuming 100% association of a-tocopheryl acetate with solids; represents maximum value. Not determined. eMass emission rates calculated as (mean concentration of a-TA in mg/L) X (mean effluent flow (L/year)]/(population) X [365 days/year); data taken from ref 3.
removal of a-TA from the F, fraction. By comparison, direct transesterification produced an apparent loss of 10.6% of the a-TA. The results of this experiment point out a methodological constraint of paramount importance to the determination of a-TA in environmental samples: hydrolytic reactions must be avoided if a-TA is to be accurately quantified. Results and Discussion Natural a-tocopherol,more commonly known as vitamin E, is the most biologically active of a family of tocopherols which occur abundantly in vegetable oils (6). Recent work was confirmed that this natural product consists of only one stereoisomer having the 2R,4’R,WR configuration (cf. Figure la). The acetate derivative of a-tocopherol apparently does not occur naturally in the marine environment, and to our knowledge its presence in uncontaminated sediments has not been reported. a-TAs are, however, produced industrially for commercial applications (e.g., nutritional supplements, cosmetics, food additives) principally because of their greater stability to oxidation than the underivitized alcohols. The two methods of acetate production most often used are (1)acetylation of a-tocopherol isolated from natural products and (2) synthesis of a-tocopherol via acidic condensation of phytol or phytyl halide (either racemic or natural) with trimethylhydroquinone followed by acetylation. The three chiral centers in a-tocopherol provide for eight possible stereoisomers. If racemic phytol is used in the synthesis, all eight stereoisomers of a-TA are present in the final (racemic) product. If natural phytol is used, only two epimers (2R,4’R,8‘R and 2S,4/R,€?R) result. Complete resolution of the diastereomers (or enantiomeric pairs) has recently been reported (7). The investiga284
Environ. Sci. Technol., Vol. 19, No. 3, 1985
tors used high-resolutiongas chromatography with a polar liquid phase after derivatization of the free alcohols. Direct chromatographic analysis of the acetate on columns coated with a nonpolar stationary phase such as DB-5, however, does not effect a separation, and all stereoisomers coelute as a single peak. Moreover, mass spectrometry is incapable of distinguishing individual stereoisomers. For these reasons, the measurements and identifications reported here reflect total a-TA content and do not constitute specific stereochemical information. In the context of this study the feature of primary importance is not the configuration of individual stereoisomers but the demonstration of the presence of the ester linkage (i.e., acetate). This is what clearly identifies the molecule as having an industrial origin. Table I lists the concentrations of a-TA measured in southern California wastewater samples along with stearic acid/a-TA ratios and per capita mass emission rates. The concentrations of a-TA in the non-sludge effluents &e., JWPCP, Hyp-Smi, OCSD, SDSD) vary over a broad range (6-25 pg/l); however, the mean annual concentrations for these effluents range only from 10 to 16 pg/L. By comparison, in sludge effluent (e.g., Hyperion 7-mile outfall, which has a higher solids content) concentrations of a-TA (in pg/L) are 40-200 times higher. If the a-TA concentrations are combined with daily effluent flow rates and population data for the treatment facilities (3,per capita mass emission rates can be computed (Table I). Values obtained in this way for southern California range from 4-14 mg/(cap-day) with a mean value of 7.5 mg/(cap-day). Data on either per capita usage rates (e.g., ingestion, topical application) or industrial production rates of a-TA within the U S . might allow an indirect means of evaluating whether our mass emission
‘T
I
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100
I50
200 250
360
GO
400
450
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M/E
L
/
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Station 3C (0-2cm) sediments F2 froction
crobial attack during and subsequent to deposition. Figure 2 shows a portion of the chromatogram of the F2 fraction isolated from the sediment sample along with a mass spectrum for the peak identified as a-tocopheryl acetate. Comparison with the mass spectrum for authentic (all racemic) a-tocopheryl acetate shown in Figure l a is conclusive proof for the presence of a-TA in these sediments. One of the major questions that remains is the extent to which a-TA can be preserved in the environment. The results of our wastewater analyses would suggest that a-TA is at least more resistant to degradation than fatty acids under the conditions of anaerobic digestion (Table I). Beyond this, little more can presently be stated with confidence as no studies on the microbial metabolism and/or physicochemical behavior of a-TA in the environment are available. It should be noted that because its anthropogenic origin is unambiguous, a-TA may prove to have a significant advantage over one of the most widely used molecular markers of domestic wastes, coprostanol. Before a-tocopheryl acetate can be confidently applied as a tracer of municipal wastes, however, more study will be needed. In particular, the ubiquity of a-TA with respect to sewage effluents must be firmly established. There is a need to know how rapidly this compound is chemically oxidized and/or decomposed by microorganisms under a variety of environmental conditions. Finally, because the ester linkage is the key structural feature linking this molecule to an industrial origin, the resistance of this bond to hydrolytic cleavage must be determined. Acknowledgments
Time Flgure 2. Partial gas chromatogram of ‘the F2 fraction Isolated from marine Sediments collected near the JWPCP waste outfall system. Electron Impact mass spectrum for the peak identified as a-tocopheryl acetate Is also shown.
rates are reasonable. Unfortunately, despite extensive efforts to secure reliable data of this type, we have been unsuccessful in doing so. The differences in n-CI8,-, acid/a-TA ratios among the effluents are striking. The anaerobically digested sludge ratios are consistently lower than those for primary effluent by factors ranging from 2.1 to 18. This suggests that a-TA has a much greater biochemical stability than fatty acids when subjected to digestion. To further explore the possibility that this compound might be used as a sewage tracer in the marine environment, we analyzed surface sediments (0-2 cm) at one site located approximately 6 km from a major submarine wastewater outfall system. The organic matter in these sediments is well characterized and has been shown to be largely of sewage origin (2,5,8,9). Therefore, one would expect to find a-TA in the sediments assuming it was sufficiently stable to survive chemical oxidation and mi-
We thank Dara L. Blumfield for laboratory assistance with the sediment samples and Ed Ruth for GC/MS analyses. Literature Cited (1) Eganhouse, R. P.; Kaplan, I. R. Enuiron. Sei. Technol. 1982, 16, 541. (2) Eganhouse, R. P.; Blumfield, D. L.; Kaplan, I. R. Enuiron. Sei. Technol. 1983, 17, 523. (3) Eganhouse, R. P.; Kaplan, I. R. Enuiron. Sci. Technol. 1982, 16, 180. (4) Eganhouse, R. P. Ph.D. Thesis, University of California, Los Angeles, CA, 1982. (5) Eganhouse, R. P.; Blumfield, D. L.; Kaplan, I. R. Proc. Int. Ocean Disposal Symp., 3rd, in press. (6) Sebrell, W. H.; Harris, R. S., Eds. “The Vitamins”, 2nd ed.; Academic Press: New York, 1972; Vol. V, Chapter 16, p 165. (7) Cohen, N.; Scott, C. G.; Neukom, C.; Lopresti, R. J.; Weber, G.; Saucy, G. Helu. Chim. Acta 1981, 64, 1158. (8) Myers, E. P. Ph.D. Thesis, California Institute of Technology, Pasadena, CA, 1974. (9) Sweeney, R. E.; Kalil, E. K.; Kaplan, I. R. Mar. Enuiron. Res. 1980, 3, 225. Received for review June 15, 1984. Accepted October 10,1984. This work was supported by the Department of Energy (Contract E Y - 76-3-03-0034).
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