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Anal. Chem. 1983, 55, 1197-1200
the molecule. The most intense metastable ion in Figure 6, (M - 55)+ at m / z 149, is probably due to elimination of 2propenal from the protonated molecular ion. That this ion is characteristic of propenal acetals is shown in Figure 3 where (M - 55)+ a t m / z 59 is by far the largest fragment in the isobutane chemical ionization mass spectrum of I. Other acetals, such as dibenzalldehydesorbitol acetal, display similar behavior in their normal and metastable isobutane chemical ionization spectra. The daughter ion spectrum of protonated I1 thus provides structural information on a minor component in the mixture, with sufficient detail to allow absolute confirmation using reference standards and with no interference from the mixture’s major components. Though not necessary in this example, the metastable transition, m / z 205 to m / z 149, may be monitored for increased specificity while following the hydrolysis behavior of I1 and its homologues in more complicated mixtures, such as polyglcyerol esters.
CONCLUSIONS Under abusive conditions, polymerization of glycerol can be accompanied by the formation of 2-propenal (acrolein). In the presence of polyols, 2-propenal leads to the formation of substituted dioxolanes. ’Volatiledioxolanes are readily isolated by headspace collection and capillary GC/ MS. Less volatile acetals, such as 11, ma:y be unambiguously identified by a powerful combination of direct mixture methods: accurate mass chemical ionization and metastable ion decomposition analysis. The hydrolysis of these acetals may be readily accomplished with dilute acid and the effectiveness of the hydrolysis may be followed by monitoring the acetal’s quasi-
molecular ions or, if necessary, the appropriate metastable ion transition. The result of the direct mixture approach is the generation of structural information that is nearly as definitive as that available from GC/MS. There are, however, two pieces of information about 2-ethenyl-4-methyl-l,3-dioxolane (I) which a r e available only through chromatography-the existence of two isomers, and the aroma characterization of the separated components via GC sniffport.
ACKNOWLEDGMENT The author is grateful to A. J. DeStefano and T. Keough for stimulating disscussions of mass spectrometry aspects of this work and to J. E. Hunter and R. P. D’Alonzo for their input concerning the chemistry of acetals. Registry No. I, 2421-07-0; 11, 85282-79-7; polyglycerol, 25618-55-7; propylene glycol, 57-55-6.
LITERATURE CITED (1) Morrison, R. T.; Boyd. R. N. “Organic Chemistry”; . Aiiyn . and Bacon: New York, 1966; p.641. Welch, R. C.; Hunter, G. L. K. J . Agric. Food Chem. 1980, 28, 870. Stenzel, W.; Franzke, C. Lebensmlffelindustrie 1977, 24 (1 I), 503. Boyd, R. K.; Beynon, J. H. Org. Mass Specfrom. 1977, 12, 163. Lacey, M. J.; Macdonald, C. G. Org. Mass Specfrom. 1977, 12, 587. Millington, D. S.; Smith, J. A. Org. Mass Specfrom. 1977, 12, 264. Stradling. R. S.; Jennings, K. R.; Evans, S. Org. Mass Specfrom. 1978, 13, 429. Veith, H. J. Org. Mass Spectrom. 1978, 13, 280. Shushan, E.; Safe, S. H.; Boyd, R. K. Anal. Chem. 1979, 51, 156. Haddon, W. F. Anal. Chem. 1970, 51, 983. Shushan, E.; Boyd, R. K. Org. Mass Specfrom. 1980, 15, 445.
RECEIVED for review October 15, 1982. Resubmitted January 12, 1983. Accepted February 4, 1983.
Extraction Efficiency of Anthracene from Sediments John D. Haddock,” Peter F. Landruni,‘ and John P. Giesy2 Savannah River Ecology Li3boratory, P.O. Drawer E, Aiken, South Carolina 2980 I
Excessive variability in the quantitation of sediment polynuclear aromatic hydrocarbon (PAH) concentrations, as revealed in interlaboratory comparisons ( I , 2), will hamper the understanding of PAH fates in the environment. The fiist problem encountered in the analysis of sediment samples for PAH is their extraction in a condition suitable for quantitation. A variety of extraction methods are currently being used and all would not be expected to be equally efficient for a matrix as complex and variable as sediment. However, appropriate corrections foir extraction efficiencies are difficult to determine. Also, it is not possible to gauge the magnitude of procedural or ~iedimentmatrix variation on extraction efficiency. The Subcomniittee on Environmental Analytical Chemistry of the American Chemical Society on Environmental Improvement has suggested that extraction efficiencies be determined by “natural incorporation” of spikes (3). Anthracene was chosen as a model PAH to assess the effects of spiking techniques, extraction solvent composition, and sediment matrix variations on extraction efficiency determinations.
EXPERIMENTAL SECTION Reagents. The reagents were of pesticide or HPLC grade. Sediment extracts were cleaned up on Florisil-PR. Chrysene Present address: Great Lakes Environmental Research Laboratory, National Oceanic and Atmospheric Administration, 2300 Washtenaw Av., Ann Arbor, MI 48104. Present address: Pesticide Research Center and Department of Fisheries and Wildlife, Michligan State University, East Lansing, MI 48824.
(Aldrich Chemical Co.) used as an internal standard was 95% pure. The [9-14C]anthracene(California Bionuclear Corp.) had an indicated specific activity of 3.3 Cismol-’ and was greater than 99% pure as determined by HPLC and liquid scintillation counting (LSC) of the eluted peeks. The scintillation cocktail was Research Products International 3a70b and the C02 trapping agent was Carbo-Sorb (Packard Instrument Co.). Sediments. Sediments were collected from two freshwater streams on the Savannah River Plant, South Carolina. Upper Three Runs Creek (UTRC) and Steel Creek (SC) sediments were collected with a glass beaker and seived through a 5 mm opening stainless steel screen. Ruhe and Matney ( 4 ) found particle size distributions for IJTRC and SC sediments of 26.1% sand, 38.4% silt, and 35.5% clay (69% kaolinite, 31% illite) for the former sediment and 85% sand, 7.5% silt, and 10.6% clay (91% kaolinite, 4% illite, and 5% chlorite-vermiculite intergrade) for the latter. Spiking Methods. All procedures were done under gold fluorescent lights. Direct sediment spiking was done just prior to extraction by adding 5 WLof an acetone stock solution containing [14C]anthracene to UTRC sediment. “Natural incorporation”of anthracene was done by mixing a slurry of wet sediment and deionized water in a 3-L, round-bottom flask with [9-14C]anthracene that had been ”plated-out” (5) of an acetone solution onto the inside of the flask by rotary-vacuum evaporation. The slurry was mixed with a magnetic stirring bar or by shaking and then vacuum filtered through Whatman No. 42 filter paper. Extraction. Spiked sediment samples to be extracted after drying were air-dried overnight in a fume hood. Spiked sediment samples to be extracted while still moist were mixed with an equal weight of anhydrous sodium sulfate. All sediment samples were Soxhlet extracted for 18 h with one of the following solvent combinations: (1)benzene; (2) acetonitrile-benzene (7:13 (v:v));
0003-2700/83/0355-1197$01.50/00 1983 American Chemical Society
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(3) acetone-cyclohexane-methanol (21:21:8 (v:v:v)). Oven dry weight of sediment was determined on sediment subsamples dried at 80 "C. Radioactivity Determinations. Carbon-14 activity was determined by liquid scintillation counting (LSC), for 10 min or to a counting error of 2% relative standard deviation, with a Beckman LS 8100 counter. Three replicate 1-mL aliquots of each solvent extract were placed in a 12 mL scintillation cocktail and counted. Preextracted and extracted sediment subsamples were analyzed for 14Cactivity after combusting and trapping the COz of 200-250 mg of sediment with a Packard Tricarb Sample Oxidizer using methods similar to Griest et al. (6). Combustion and recovery efficiencies were determined by comparing the activity of an external 14Chydrocarbon standard to the recovered activity of combusted standard. Disintegrations per minute were calculated for all samples by correcting the measured activities for background and counting efficiency with a standard quench curve based on the sample channels ratio method. High-pressure Liquid Chromatography. Extracts selected for HPLC analysis for determination of anthracene degradation were from UTRC and SC sediments that had been slurry spiked for 1 and 8 days and extracted while moist with acetonitrilebenzene. The extracts were rotary-vacuumevaporated to -2 mL and passed through a 7 mm i.d. X 12 cm glass cleanup column containing -0.8 g of benzene rinsed Florisil-PR. The columns were eluted with 25 mL each of a methylene chloride-benzene solution (1:9 (v:v)). The eluant was collected and rotary-vacuum and nitrogen stream evaporated to 250 pL. Two milliliters of acetonitrile containing chrysene as an internal standard was then added to each sample followed by vortex mixing. Each sample was then centrifuged for 10 min at 2000 rpm and analyzed on a Varian 5000 high-pressure liquid chromatograph equipped with a 254-nm fixed wavelength absorbance detector. Automatic injections with a 25-hL loop were made on a Varian 4 mm i.d. X 30 cm, C-18, reversed-phase column. The mobile phase composition was gradient increased linearly in 40 min, from 35% acetonitrile in water to 100% acetonitrile with a 10 min hold at 100% acetonitrile, and then returned to initial conditions. The mobile phase flow rate was 1 mLernin-l. The eluting peaks were collected for LSC immediately after exiting the detector. Experimental Design. Three replicate sediment samples per treatment were used in all experiments. The first experiment, with UTRC sediment, compared the extraction recoveries of [14C]anthracenefrom direct vs. 18 h aqueous slurry-spiked sediment. After filtration, 8 g dry weight each of moist UTRC sediment was directly spiked with 1.5 f 0.0054 pgg-l (R f std dev) [ 14C]anthracene and Soxhlet extracted with acetonitrilebenzene. The slurry-spiked sediment samples weighed approximately 3.5 g dry weight each with a [14C]anthraceneconcentration of 3.09 f 0.026 pg-g-l and were extracted with acetonitrile-benzene (7:13 (v:v)) or acetone-cyclohexane-methanol (21:21:8 (v:v:v)). The second experiment employed a Z4 complete factorial experimental design to evaluate the effects of four variables on anthracene extraction efficienciesof slurry-spiked sediment. The variables were (1) sediment type, UTRC or SC, (2) mixing time of the sediment slurry plus [14C]anthracenespike, 1 or 8 days, (3) sediment moisture prior to extraction, vacuum filtered and mixed with sodium sulfate (wet) or air-dried, (4) extraction solvent composition, acetonitrile-benzene (713 (KV)) or benzene. Nominal [14C]anthracenesediment concentration was 1.0 wg-g-' dry weight. Sediment sample size was approximately 3 g oven dry weight. Extraction efficiencies were calculated as the ratio of extracted 14C activity and preextraction I4C activity. Preextraction 14C activity was determiened by combustion and LSC of spiked sediment samples. Mass balance checks were done for the UTRC and SC sediments, slurry spiked for 1 day, air-dried, and extracted with acetonitrile-benzene treatment combinations, by combustion of extracted sediment to determine unextracted 14C activity. Calibration of this technique established a combustion and trapping efficiency of 93% for a 14C hydrocarbon standard. Statistical calculations were made with the Statistical Analysis System (7).
RESULTS A N D DISCUSSION Anthracene spiked directly into UTRC sediment samples just prior to extraction with acetonitrile-benzene was almost
Table I. Extraction of [14C]Anthracenefrom Direct and Slurry Spiked UTRC Sediment treatment
solvent systema
extraction efficiency,c %
direct spike slurry spike slurry spike
AB (7:13 ( v : ~ ) ) ~ AB ( 7 : 1 3 (v:v)) AtCM (21:21:8 (v:v:v))
97.6 f 5 . 0 80.8 f 7.5 89.8 f 6.7
a A = acetonitrile, B = benzene, C = cyclohexane, At =acetone, M = methanol. Volume ratio of solvents. e X 95%CI, n = 3.
Table 11. Concentrations of [I4C]Anthracene in Slurry Spiked Sediments concentration spike sediment . time, moisng-g-' 95% sedimenta days tureb (dry wt) CI UTRC
sc
1 1 8 8 1 1
8 8
wet dry wet dry wet dry wet dry
846.1 899.3 900.0 912.7 647.9 567.9 560.1 533.1
71.5 10.0 25.3 6.8 34.8 29.9 30.7 17.3
f
n 5 23 6 24 6 24 5 24
UTRC = Upper Three Runs Creek, SC = Steel Creek. Wet sediment was assayed for I4C activity after vacuum filtration, while dry refers to vacuum filtered sediment that was air-dried for 24 h. a
quantitatively recovered, while slurry-spiked sediment gave significantly lower recoveries (Duncan's multiple range test, a = 0.05) (DMRT) (Table I). Absence of the acetone carrier solvent and greater mixing with the latter technique more closely simulated natural sorption processes than the former and may have decreased extraction efficiency. The acetone-cyclohexane-methanol solution, which constituted a ternary azeotrophic system (8) was the most efficient solvent system for the extraction of anthracene from slurry-spiked sediment, although it coextracted additional material that precipitated out of solution during volume reduction of the extracts which interfered with subsequent analyses. The acetonitrile-benzene azeotropic system (8)was significantly less efficient (DMRT) than the ternary system but was more practical since it coextracted less interfering material. In a second experiment in which the slurry-spiking technique only was used, UTRC sediment sorbed more of the spike than did SC sediment (Table 11). This was probably due to the greater fractions of organic carbon and clay in UTRC sediment than in SC sediment. Ruhe and Matney ( 4 ) found organic carbon contents of 18.9 and 7.1% for UTRC and SC sediments, respectively. Sorption of hydrophobic compounds by sediments has been shown to be highly correlated with the organic carbon content of sediments (5, 9). The amount of anthracene sorbed by both sediments was not increased by mixing for 8 days; and, air-drying spiked SC sediment for 24 h may have slightly reduced the anthracene concentration through volatilization (Table 11). Extraction efficiency of anthracene from the sediments was dependent on the treatment and ranged from 30.9 4.0 to 97.2 f 8.7% (Xf 95% CI) (Table 111). Of the four variables examined, mixing duration of spiked slurries was the most significant factor affecting extraction efficiency (Tables I11 and IV). In pairwise comparisons between treatment means, recovery was always significantly greater (DMRT) from sediments mixed for 1 day than from 8 day mixed sediments, except for the 1 and 8 day mixed, air-dried, benzene-extracted sediments from both UTRC and SC (Table 111). Mass bal-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
Table 111. Recovery of [I4C]Anthracene from Slurry Spiked Sediment spike sediment solvent time, mois- composedimenta days ture sitionb UTRC
1
dry wet
8
dry wet
AB B AB B AB B AB
B
sc
1
dry wet
8
dry wet
AB B AB B AB B AB B
UTRC
85.7 * 2.7 81.6 f 6 . 2 78.4 f 15.2 79.7 f 9.9 73.0 i 1.0 70.9 f 9.4 47.0 i 8.7 32.8 (n = 2 ) d 80.7 f 7.5 51.8 f 13.4 87.8 f 6 . 2 97.2 f 8.7 54.9 i: 2.5 46.9 f 0.75 53.7 f 7.7 30.9 f 4.0
sc
UTRC = Upper Three Runs Creek, SC = Steel Creek. AB = acetonitrile-benzene, B = benzene. 2 i 96%CI, n = 3, except where noted. One value omitted, Chauvenet's criterion ( I 0). Table IV. Analysis of Variance of [I4C]Anthracene Recoveries from Sediments source
df
sediment ( A ) spike time (B) moisture (C) solvent ( D ) A, B A, c A, D B, C B, D C, D A, B, C A, B, D A, c , D B, c, D A, B, C, D error total a
P 6 0.05.
1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 31 46
P 6 0.01.
Table V. Percentage of 14C Activity Found in the HPLC Anthracene Peak sediment a spike time, days [Wlanthrance, %
recovery,' %
a
mean square
F
0.03696 0.98418 0.02638 0.08770 0.01601 0.21451 0.017 58 0.28272 0.011 26 0.00515 0.00392 0.00009 0.01662 0.09068 0.02266 0.00104
35.5' 945.7' 25.4' 84.3' 15.4' 206.1' 16.9' 271.7' 10.8 5.0" 3.8; 0.1 16.0' 87.1' 21.8'
P < 0.001. Not significant.
ances (extracted + unextracted 14Cactivity) for UTRC and SC sediments spiked for 1 day and extracted with acetonitrile-benzene after air-drying were 99.1 f 0.2% and 104.4 f 8.2% (8f std dev, n = 3), respectively. This indicates that anthracene was not lost through volatilization during the extraction step. Anthracene degradation as determined by HPLC and LSC of collected peaks showed some degradation to more polar compounds, but greater than 85% of the 14C activity was associated with the anthracene peak (Table V). Since degradation was similar for both sediments and did not increase with miming tiime, it probably occurred after extraction during storage of the extrads prior to HPLC analysis, which would not have influenced extraction efficiency. The decrease in extraction efficiencies with increased contact time between sediment and anthracene during mixing may have involved increased partitioning or movement of anthracene to interclay lattices. Kisrickhoff (11) observed decreasing extraction efficiencies f a r naphthalene, phenanthrene, and pyrene with increasing iincubation time of these PAH compounds and sediment and suggested a relationship to diffusive transfer of the compounds on or into sediment particles. Herbes (12) found that the percentage of unextractable
I199
1 8 1 8
93.6 i 2.6 88.8 f 1.8 86.6 i 2.8 87.4 f 4.4
a-UTRC = Upper 'Three Runs Creek, SC = Steel Creek. b X f 95%CI,n=3. ("bound") [ 14C]anthracene increased over a 25-h incubation period for a stream sediment. Extracting solvent composition, sediment type, and sediment moisture also significantly affected extraction efficiency (Table IV). However, their contribution to the overall variability was slight in comparison with the sediment-anthracene mixing time or with the variable interactions (Table IV). General trends in extraction efficiencies as shown by the main variable means were as follows: for sediment type, UTRC > SC; for solvent composition, acetonitrile-benzene > benzene; and for sediment mositure, air-dried > wet. A notable exception was for 1 day mixed, SC sediment extracted with benzene, in which extraction of the wet sediment gave a 45.4% higher recovery over dry sediment (Table 111). This result, which is unusual since benzene is immiscible with water and should be less efficient for extraction of wet sediment than for dry sediment, is accounted for by the significant interactions among the variables (Table IV). There were significant interactions among the variables for all but two treatment combinations (Table IV). Taken together, the significant interaction sum of squares accounted for 37.7% of the total sum of squares. This indicates that the independent variables affected each other in a nonadditive manner, and accurate predictions of extraction efficiencies based on knowledge of the main effects alone cannot be made. Extraction efficiencies are important for evaluating extraction methods as well as for correction of compound concentrations in sediment samples. Also, when variables are manipulated in a controlled manner, they provide information regarding the disposition of compounds sorbed by sediments. The relative importance of sediment components involved in sorption can be determined by alteration of their concentration or composition or by choice of extracting solvents that are selective in their interaction with those components. The difficulty of determing accurate extraction efficiencies for PAH from sediments has been reflected in the literature by the varieity of approaches taken (13-16). However, the time and expense as well as the importance of quantifying PAH and other trace environmental pollutants necessitate the effort. We have shown significant variability in the extraction efficiency of anthracene from sediment due to spiking technique and extraction method variables and their interactions. Studies designed to investigate additional variables and compounds will contribute to the understanding of the environmental fate of trace organic compounds in the environment by providing a broader basis for comparison of measurements made by different laboratories using different techniques. Ultimately, a standardization of methodology may be possible.
ACKNOWLEDGMENT We appreciate the laboratory and statistical assistance of Karen Brown. Also, thanks go to John Pinder for help with experimental design and for helpful suggestions offered by R. Zepp and D. Paris on analytical techniques. The comments of M. Zabik, M. Snook, R. Arrendale, J. Alberts, J. Pinder, and J. Bowling on initial drafts of this manuscript were helpful. P a t Davis typed the manuscript.
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Registry No. Anthracene, 120-12-7;benzene, 71-43-2; acetonitrile, 75-05-8;acetone, 67-64-1;cyclohexane, 110-82-7;methanol, 67-56-1.
LITERATURE CITED (1) Hilpert, L. R.; May, W. E.; Wise, S. A.; Cheder, S. N.; Hertz, H. S. Anal. Chem. 1978, 50, 458-463. (2) MacLeod, W. D., Jr.; Prohaska, P. G.; Gennero, D. D.; Brown, D. W. Anal. Chem. 1982, 54, 386-392. (3) MacDougall, D.; Crummett, W. Anal. Chem. 1980, 52, 2242-2249. (4) Ruhe, R. V.; Matney, E. A. "Clay Mlneralogy of Selected Sedlments and Soils at the Savannah River Plant, Alken, South Carolina", DP-MS80-119; Savannah River Laboratory: Alken, SC, 1980. (5) Karickhoff, S. W.; Brown, D. S.; Scott, T. A. Wafer Res. 1979, 13, 24 1-248. (6) Griest, W. H.; Yeatts, L. B., Jr.; Caton, J. E. Anal. Chem. 1980, 52, 199-201. (7) Statistical Analysis System; SAS Institute Inc.: Raleigh, NC, 1979. (8) Weast, R. C., Ed. "Handbook of Chemistry and Physics", 61st ed.; CRC Press: Boca Raton, FL, 1980. (9) Means, J. C.; Hassett, J. J.; Wood, S. 0.;Banwart, W. L. "Polynuclear Aromatic Hydrocarbons"; Jones, P. W., Leber, P., Eds.; Ann Arbor Science: Ann Arbor, MI, 1979.
(IO) Chase, 0. D.; Rablnowltz, J. L. "Principles of Radioisotope Methodology", 3rd ed.; Burgess: Mlnneapolis, MN, 1967; Chapter 4. (1 1) Karlckhoff, S. W. "Contaminants and Sediments"; Baker, R. A., Ed.; Ann Arbor Science: Ann Arbor, MI, 1980; Chapter 11, pp 193-205. (12) Herbes, S. E. Appl. Envlron. Mlcrobiol. 1981, 41, 20-28. (13) Brown, R. A.; Starnes, P. K. Mar. Pollut. Bull. 1978, 9 , 162-165. (14) Dunn, B. P. Envlron. Scl. Techno/. 1978, 10, 1018-1021. (15) Cretney, W. J.; Christensen. P. A.; McIntyre, 8. W.; Fowler, B. R. Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment": Afahan. B. K.. Mackav. D.. Eds.: Plenum: New York. 1980; pp 315-3316. (16) Lake, J. L.; Dlmock, C. W.; Norwood, C. B. Adv. Chem. Ser. 1980, No. 185 (Pet. Mar. Envlron.), 343-360.
RECEIVED for review October 20, 1982. Accepted February 16,1983. This study was supported by interagency agreement EPA-79-D-X0533 between the U.S. Department of Energy and the U.S. Environmental Protection Agency and Contract DE-AC09-76R00819between the U S . Department of Energy and the University of Georgia.
