14 Ambient-Temperature Extraction of Hydrocarbons From Marine SedimentComparison with Boiling-Solvent Extractions DONALD W. BROWN, L. SCOTT RAMOS, MARIANNE Y. UYEDA, ANDREW J. FRIEDMAN, and WILLIAM D. MACLEOD, JR. 1
NOAA National Analytical Facility, Environmental Conservation Division, Northwest and Alaska Fisheries Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, WA 98112
Effective measurement of hydrocarbons in marine sediments requires standardized extraction procedures that are efficient and reproducible. Most hydrocarbon extractions from sediment depend on solvent reflux, which poses difficulties for routine processing of large numbers of samples. As an alternative, an ambient-temperature solvent extraction was devised for a ball-mill tumbler and compared with three boiling-solvent extractions. Aliphatic and aromatic hydrocarbons from a moderately contaminated sediment were quantitated using glass capillary gas chromatography. After dewatering the sediment with methanol, the tumbler extraction with 2:1 dichloromethane:methanol gave hydrocarbon yields comparable to the boiling-solvent extractions. The tumbler extraction did not require benzene, hood space, expensive glassware, boiling solvents, running water, or freeze-drying.
/ C o n c e r n over oil pollution has led to considerable interest i n measuring ^ amounts of hydrocarbons i n the marine environment ( 1 , 2 ) . A number of researchers have developed specialized procedures for analyzing hydrocarbons i n marine sediments (3-14). Unfortunately, because 1
Author to whom reprint requests should be sent. This chapter not subject to U.S. copyright. Published 1980 American Chemical Society
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the various procedures have not been adequately assessed (3,4), inter pretations of analytical data from these analyses have been difficult, especially for individual aliphatic and aromatic hydrocarbons. To help overcome such deficiencies, it is desirable to compare important analytical methods for hydrocarbons i n marine sediment using a representative set of individual hydrocarbons. Clearly, the extraction techniques compared should be reproducible, accurate, and efficient within meaningful limits. In addition, these techniques must be safe and convenient for processing large numbers of samples. Soxhlet extraction with benzene and methanol (3,5,9,10,14) is generally considered the most efficient technique for hydrocarbon re covery. However, it is not convenient for processing large numbers of samples ( β ) . Recently, alternative techniques have been reported that may be as efficient as soxhlet extraction. F o r example, Farrington and Tripp (5) showed that sediment refluxed for 3 hr w i t h benzene and methanol afforded about the same gross weight of hydrocarbons as by the soxhlet technique. Similarly, Rohrback and Reed (3) reported that by shaking sediment with various solvents, they extracted almost the same weight of hydrocarbons as by soxhlet extraction with the same solvents. However, M a c L e o d et al. (6) often found that shaking pro duced stable emulsions. T o avoid emulsions, Warner suggested extracting sediments with diethyl ether-water using a ball-mill tumbler (8). This approach reduced emulsions and was convenient for mass sample process ing (6,7), but Carpenter and Bates found that this technique generally extracted only about one-third the amount of hydrocarbons extracted by the conventional soxhlet technique (9). W e have derived an extraction method that retains the convenience of the ball-mill tumbler for large numbers of extractions, and have investigated various solvent systems for improved recoveries. Preliminary results indicate that a tumbler extraction using methanol and dichloromethane recovered hydrocarbons from an intertidal sediment as efficiently as the soxhlet technique (12). This report describes improvements i n our tumbler procedure and compares it with three boiling-solvent pro cedures (10,13,15). Materials
and Methods
Materials, reagents, apparatus, and their cleaning procedures have been published previously (6,12). A l l solvent ratios were on a v / v basis. Sediment dry weights were determined on 10-20-g samples (6). Methanol Purification. T o check methanol purity, 200 m L was diluted with an equal volume of contaminant-free dichloromethane and extracted twice w i t h 200-mL portions of distilled water. After the resulting dichloromethane phase was concentrated to 1 m L , it was
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315
analyzed for contaminants by glass capillary gas chromatography ( G C ) . W h e n contaminants exceeded 0.1 n g / ^ L i n the dichloromethane concen trate (i.e., 0.5 / x g / L methanol), 1500 m L of the methanol was purified by diluting it with 500 m L of water and extracting it twice with 50-mL aliquots of contaminant-free hexane. Then the aqueous methanol was redistilled at 65°-67°C through an efficient fractionation column and checked by G C analysis. Sediment Extraction Procedures. B A L L - M I L L T U M B L E R E X T R A C TION. Figure 1 shows the extraction scheme. A 100-g sample of wet sediment was weighed into a 1-L bottle, and 50 m L of methanol were added. Aliquots of recovery standards ( n-decylcyclohexane and 1,3,5triisopropylbenzene) were added to each sample, except for the reagent blank. A n aliquot containing the compounds to be quantitated was added to a second blank to estimate typical losses due to handling. The bottles containing the samples and methanol were gently swirled by hand to dewater the sediment. The methanol was decanted into a 600-mL beaker, and the methanol-dewatering step was repeated with another 50 m L of methanol. Then 100 m L of 2:1 dichloromethane:methanol was added to the sediment, and the bottles were sealed with all-Teflon screwcaps and rolled on a ball-mill tumbler for 16 hr (overnight) at about 75 rpm. The extract was decanted into the 600-mL beaker containing the methanolic extracts. Then the sample and bottle were washed w i t h about 5 m L of dichloromethane (dispensed from a clean Teflon wash bottle), and the washings were decanted into the 600-mL beaker. The dichloromethane-methanol sediment extraction step was repeated twice, first for 6 hr, then for 16 hr. A l l extracts were combined and filtered through a 65-mm-i.d., coarse fritted-glass filter funnel into a 1-L separatory funnel. The beaker and filter were washed twice with 25-50 m L of dichloromethane. The total filtrate was gently swirled for 2 min with 500 m L of distilled water to remove methanol from the dichloromethane phase. After the phases separated, the dichloromethane (lower) phase was drained into a 500-mL Erlenmeyer flask. The aqueous (upper) phase was then back-extracted with 20 m L of dichloromethane and the dichloromethane phases were combined. The aqueous phase was discarded. The aqueous wash-dichloromethane back-extraction steps were repeated on the combined dichloromethane extracts prior to cleanup (below). D I R E C T R E F L U X E X T R A C T I O N . Wet sediments (50 g ) were refluxed in 250 m L of 0.5N K O H in methanol and 35 m L of distilled water for 2 hr (IS). After cooling, the mixture was poured through a 65-mm-i.d., coarse fritted-glass filter funnel into a 1-L separatory funnel. The extrac tion flask and the filter were rinsed with 20 m L of methanol and three times with 35 m L of dichloromethane. Distilled water (150 m L ) was added to the extract and the mixture was shaken. After the phases separated, the dichloromethane layer was drained into a 500-mL E r l e n meyer flask, and the aqueous phase was back-extracted with 50 m L of dichloromethane. The combined dichloromethane extracts then passed to the cleanup step (below). S O X H L E T E X T R A C T I O N W I T H Β Ε Ν Ζ Ε Ν Ε - M E T H A N O L . The procedure of Clark and Finley (10) was employed, using two 24-hr extractions of a 100-g sample of wet sediment, each with 250 m L of 1:1 benzene:methanol.
316
PETROLEUM
WET
ENVIRONMENT
SEDIMENT
Wash w i t h
CH 0H/H 0 3
IN T H E MARINE
CH 0H 3
DEWATERED SEDIMENT
Γ
2
EXTRACT
E x t r a c t on t u m b l e r w i t h 2:1 CH C1 /CH 0H 2
2
3
EXTRACTED SEDIMENT
CH C1 /CH 0H 2
2
3
Ί
EXTRACT
• Discard
"Combine
TOTAL
EXTRACT
I WASHED
CH C1 2
JU
2
••Discard
EXTRACT
Filter
through s i l i c a g e l precolumn
I PARTIALLY PURIFIED CH C1 EXTRACT 2
2
C o n c e n t r a t e and d i s p l a c e CH C1 with " ~ C H 2
2
6
1 4
CONCENTRATED EXTRACT IN n - C H 6
u
Chromatographic s e p a r a t i o n with
1
SATURATED HYDROCARBONS
Glass
Capillary
t
S i l i c a Gel
AROMATIC HYDROCARBONS
Gas C h r o m a t o g r a p h y
GC/MS C o n f i r m a t i o n s
Figure 1.
Ball-mill tumbler extraction scheme
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317
Extractions of Hydrocarbons from Sediments
A L .
The combined extracts were washed with water, dried over N a S 0 , and evaporated just to dryness w i t h a rotary evaporator. The residue was dissolved in dichloromethane for extract cleanup (below). 2
S O X H L E T
E X T R A C T I O N
W I T H
D I C H I X ) R O M E T H A N E - M E T H A N O L .
4
Wet
sediments (100 g) were extracted by a similar soxhlet procedure using 2:1 methanol:dichloromethane (15). The extracts were combined, washed, and concentrated as done in the tumbler procedure prior to cleanup. Extract Cleanup. The sediment extract i n dichloromethane was filtered through a 19-mm-i.d. chromatography column containing 20 m L of activated silica gel previously prepared in dichloromethane and covered with a 1-cm layer of sand (6). Then the column was eluted with one bed volume of dichloromethane. The combined eluates were collected in a 500-mL Erlenmeyer flask equipped with a 24/40 outer joint. Residual methanol, water, particulates, and gel-forming polar materials (which could plug the silica gel chromatography column used later) were removed by this step. A Snyder distillation column equipped w i t h a 24/40 inner joint was attached to the flask, and the assembly was placed in a heated (ca. 60°C) water bath. The dichloromethane extract and eluate were concentrated to ca. 15 m L and transferred to a 25-mL Kontes concentrator tube. After a Teflon boiling chip was added, and a Kontes micro-Snyder column (modified w i t h indentations) was attached, the extract was further concentrated on a modified Kontes tube heater to about 1 m L . After adding 2 m L of hexane, the extract was reconcentrated to about 1 m L for fractionation into hydrocarbon classes. Fractionation into Hydrocarbon Classes. A l l extracts were chromatographed on Davison grade 923 silica gel, as reported earlier ( 6 , 7 ) . Two fractions, containing saturated and unsaturated hydrocarbons, re spectively, were collected i n separate 25-mL Kontes concentrator tubes. These fractions were concentrated to 1 m L on a modified Kontes tube heater. After adding 2 m L of hexane, the extract was reconcentrated to 1 m L and transferred to G C sample vials. After adding 4 μg of hexamethylbenzene ( G C internal standard) in hexane, the vials were sealed for G C analysis. Gas Chromatographic Analysis. The vial contents were automati cally sampled and analyzed by G C (6,7) using high-resolution glass capillary columns (see Figure 2 for column parameters and operating conditions). Major alkanes ranging from decane ( η - Ο ι Η ) through hentriacontane ( n - C i H ) , plus pristane and phytane, were quantitated in the saturated hydrocarbon fraction. The arenes listed i n Table I were quantitated in the unsaturated hydrocarbon fraction. 0
3
Results and
22
6 4
Discussion
Ball-Mill Tumbler Extraction. T o avoid unnecessary cost, incon venience, and hazards involved i n extracting large numbers of sediment samples with boiling solvents (6), we have investigated alternative pro cedures. Ideally, a suitable procedure should extract efficiently and reproducibly and be simple, safe, and convenient. I n particular, we needed a method that used minimal benchtop or hood space; could
Figure 2A. Gas chromatograms of the saturated hydrocarbons from an extract of lower Duwamish River sediment, West Waterway, Seattle, Washington, Hewlett-Packard 5840A GC with flame ionization detector. J ÙW Scientific 30 m X 0.25-mm SE-54 WCOT column obtained from Supelco. 2μΕ splitless injection, vaporization at 280°C, split 10:1 after 18 sec with 22 psig helium carrier gas. Column temperature 40°C for 5 min, then programmed to 270°C at 4°/min. Numbered peaks refer to n-alkanes of corresponding chain length.
CO
ι
00
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Extractions of Hydrocarbons from Sediments
v. ft) auaj*d(e)ozuag
|8 H co
S*
ν. § g •"•s "β J Sa"
§§
ni ο § α
co "g
SbtS 6
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. 8
319
320
PETROLEUM
Table I.
IN T H E M A R I N E E N V I R O N M E N T
Concentrations of Aromatic Hydrocarbons Extraction CH Cl -CH OH 2
Aromatic
Hydrocarbon
2-Methylnaphthalene 1 -Methylnaphthalene Biphenyl 2,6-Dimethylnaphthalene 2,3,5-Trimethylnaphthalene Fluorene Dibenzothiophene * Phenanthrene Anthracene 1-Methylphenanthrene Fluoranthene Pyrene Benzanthracene Chrysene Benzofe] pyrene Benzo[a]pyrene Perylene
2
s
Tumble
(ng/g)
RSD (%)
10 6 2 8 6 30 28 330 57 22 570 760 440 270 150 170 36
33 33 39 26 58 28 32 28 26 24 23 21 23 20 26 33 36
Data reported as ng/g dry weight; χ = mean, η — number of analyses, R S D relative standard deviation of the mean (100 SD/af). a
=
function at ambient temperature; avoided freeze-drying; and avoided benzene, a suspected carcinogen. After more than a years experience with solvent-slurry extractions of sediments using a ball-mill tumbler (β, 7 ) , we devised a procedure using methanol and dichloromethane that largely met these criteria. In developing this tumbling extraction procedure, a number of solvent systems were investigated, including: Diethyl ether-acidified water Dichloromethane-acidified water Dichloromethane : diethyl ether (2:1) -acidified water Diethyl ether:methanol (2:1) Dichloromethane:methanol (2:1), (1:1), and (1:2) Comparison of these tumbler solvent systems showed that if methanol was one of the solvents, hydrocarbon extraction efficiencies were 2-3 times better than without it, regardless of the less polar cosolvent used. Hence, methanol was chosen as a cosolvent for use with dichloromethane, which is safer than diethyl ether or benzene. To optimize our tumbler method, we tested various ratios of dichloro methane to methanol at various solvent-to-sediment ratios. Replicate
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321
Found in Homogenized Duwamish River Sediment by Four Methods α
Soxhlet CH OH-KOH s
x(n = 4) (ng/g) 7
4 1 5 3 3 % ). Usually, direct reflux extraction was least efficient but most reproducible Table II.
Concentrations of Aliphatic Hydrocarbons Extraction CH Cl -CH OH s
Tumble
x(n = 14) (ng/g)
RSD (%)
2
n-Alkane
Pristane* Ois Phytane 6
2
6 11 18 23 36 51 44 39 54 40 28 29 39 36 52 43 51 54 72 98 96
Data reported as ng/g dry weight; χ = mean, η = relative standard deviation of the mean (100 SD/x). β
23 25 19 26 16 18 15 16 15 14 24 14 15 15 15 23 32 33 30 35 55 number of analyses; R S D
=
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Extractions of Hydrocarbons from Sediments
323
for both alkanes and aromatics. Recoveries for the polynuclear aromatics below fluorene i n Table I were generally highest by benzene-methanol soxhlet extraction. Again dichloromethane-methanol soxhlet extraction was least reproducible (highest R S D s ) . Variability of Aromatic Hydrocarbons. Figure 2B shows that the unsubstituted aromatics are generally more abundant than their alkylsubstituted homologues. Recent research of L a F l a m m e and Hites (16) and Youngblood and Blumer (17) indicates that this pattern is character istic of combustion byproducts as opposed to spilled fossil fuels. If these aromatics had such an origin and were deposited with various types of airborne particulates, they could give uneven results for this sediment, even though it was homogenized i n a mixer for 3 hr. This is consistent with the greater variability observed for the aromatic data compared w i t h the alkane data (e.g., 28 out of 68 R S D s > 3 3 % i n Table I vs. 10 out of 84 RSDs > 3 3 % i n Table I I ) . Found in Homogenized Duwamish River Sediment by Four Methods* Soxhlet C H OH-KOH s
Reflux
Extraction
Benzene-CH OH
CH Cl -CH OH
3
2
2
3
x(n = 5) (ng/g)
RSD (%)
χ (n = 5) (ng/g)
RSD (%)
(ng/g)
RSD (%)
4 8 15 20 29 40 28 33 31 30 35 22 27 31 37 34 59 45 42 36 36
17 16 12 10 7 8 9 7 5 9 12 8 7 5 9 11 15 11 11 15 10
6 11 18 23 29 40 39 33 41 38 39 30 34 35 44 47 62 110 100 98 144
14 15 16 16 24 42 17 29 33 20 15 15 12 13 8 15 23 15 17 20 23
5 9 15 20 29 37 33 32 32 28 36 30 40 46 74 69 150 170 160 180 130
32 29 29 24 15 15 22 15 7 4 4 7 20 20 51 48 63 57 46 120 87
* A branched alkane.
324
PETROLEUM
IN T H E M A R I N E E N V I R O N M E N T
Figure 3. Gas chromatograms of 200-fold concentrated extracts of (A) high-quality, commercially available methanol, (B) the same methanol after fractional redistillation, and (C) the same methanol purified by dilution with water, extraction with hexane, and fractional redistillation. GC parameters and conditions same as in Figure 2.
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In the extreme, the overall recoveries of aromatics in 5 of 28 analyses exceeded the overall mean of their respective subsets (n > 5) by factors of 3, 3, 5, and 10, while the corresponding alkane recoveries d i d not. These exceptional step increases in only the aromatics may be due to the presence of one or more aromatic-rich particles (e.g., soot). This possibility is not excluded since the sediment came from an active harbor in an urban industrial area. Dixon's statistical method of outlier analysis (18) was used to exclude such extreme results from Tables I and II. Impurities in Methanol. W e found that methanol was an impor tant cosolvent for extracting hydrocarbons efficiently from the wet sedi ment. E v e n the purest of more than 10 top grades of commercial methanol had impurities that interfered w i t h hydrocarbon analyses ( Figure 3 A ). Attempts to purify such methanol by fractional redistillation removed only contaminants boiling higher than n - C i H G (Figure 3 B ) . To reduce contaminants less volatile than n - C H i to acceptable levels ( < 0.5 μg/L·), methanol was diluted with water, extracted with hexane, and redistilled ( Figure 3 C ). 2
8
2
S
Summary W e developed a hydrocarbon extraction procedure for sediments that uses methanol and dichloromethane with a ball-mill tumbler. W i t h i n the limits of experimental error ( 1 σ ) , this procedure is generally as efficient as soxhlet extraction with benzene—methanol or dichloromethane-methanol and more efficient than direct reflux with alkaline methanol. The new procedure, which is convenient for processing large numbers of samples, uses less expensive equipment and less toxic solvents than previously employed soxhlet techniques. Ambient-temperature extraction eliminates the problems inherent with boiling solvents and cooling water. Moreover, dewatering with methanol makes freeze drying or air drying unnecessary. Acknowledgments This study was supported by the Bureau of L a n d Management through the Outer Continental Shelf Environmental Assessment Program ( O C S E A P ) of the National Oceanic and Atmospheric Administration ( N O A A ) . W e thank J . A . Calder and J . J . Kineman of O C S E A P for 50 k g of homogenized sediment; D . D . Gennero, P. G . Prohaska, D . D . Dungan, and J . S. Finley for laboratory assistance; R. G . Jenkins and T. I. Sherman for data preparation; and F . J . Ossiander for statistical evaluation. Reference to a company or a product does not imply endorse ment by the U.S. Department of Commerce to the exclusion of others that may be suitable.
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Literature Cited 1. Clark, R. C., Jr.; Brown, D. W. "Petroleum: Properties and Analyses in Biotic and Abiotic Systems," in "Effects of Petroleum on Arctic and Subarctic Marine Environments and Organisms, Nature and Fate of Petroleum"; Malins, D. C., Ed.; Academic: New York, 1977; Vol. I, pp. 1-89. 2. Farrington, J. W.; Teal, J. M.; Parker, P. L . "Petroleum Hydrocarbons," in "Strategies for Marine Pollution Monitoring"; Goldberg, E . D., Ed.; John Wiley: New York, 1976;pp.3-34. 3. Rohrback, B. G.; Reed, W. E . "Evaluation of Extraction Techniques for Hydrocarbons in Marine Sediments," Institute of Geophysics and Plane tary Physics, University of California, Los Angeles, CA, 1975, No. 1537. 4. Hilpert, L . R.; May, W. E.; Wise, S. Α.; Chesler, S. N.; Hertz, H. S. "Interlaboratory Comparison of Determinations of Trace Level Petroleum Hydrocarbons in Marine Sediments," Anal. Chem. 1978, 50, 458. 5. Farrington, J. W.; Tripp, B. W. in "Marine Chemistry in the Coastal Envi ronment," ACS Symp. Ser. 1975,18,267-284. 6. MacLeod, W. D.; Brown, D. W.; Jenkins, R.G.;Ramos, L. S.; Henry, V. D. "A Pilot Study on the Design of a Petroleum Hydrocarbon Baseline In vestigation for Northern Puget Sound and Strait of Juan de Fuca," NOAA Tech. Memo. ERL MESA-8, National Oceanic and Atmospheric Administration, Boulder, CO, 1977. 7. MacLeod, W. D.; Brown, D. W.; Jenkins, R. G.; Ramos, L. S. "Intertidal Sediment Hydrocarbon Levels at Two Sites on the Strait of Juan de Fuca," In "Fate and Effects of Petroleum Hydrocarbons in Marine Or ganisms and Ecosystems"; Wilfe, D. Α., Ed.; Pergamon: New York, 1977; pp. 385-396. 8. Warner, J. S., Battelle Memorial Institute, Columbus, OH, personal com munication, 1976. 9. Carpenter, R.; Bates, T., Department of Oceanography, University of Washington, Seattle, WA, personal communication, 1977. 10. Clark, R. C., Jr.; Finley, J. S. "Techniques for Analysis of Paraffin Hydro carbons and for Interpretation of Data to Assess Oil Spill Effects in Aquatic Organisms,"Proc.Jt. Conf. Prev. Control Oil Spills 1973, 161172. 11. Chesler, S. N . Gump, B. H.; Hertz, H . S.; May, W. E.; Dyszel, S. M.; Enaganio, D. P. "Trace Hydrocarbon Analysis: The National Bureau of Standards Prince William Sound/Northeastern Gulf of Alaska Baseline Study," Nat. Bur. Stand. (U.S.), Tech. Note 1976, 889. 12. Brown, D. W.; Ramos, L. S.; Friedman, A. J.; MacLeod, W. D. "Analysis of Trace Levels of Petroleum Hydrocarbons in Marine Sediments Using a Solvent Slurry Extraction Procedure," In "Proceedings 9th Materials Symposium, Trace Organic Analysis: A New Frontier in Analytical Chemistry"; National Bureau of Standards: Washington, DC, 1979. 13. Quinn, James G., Graduate School of Oceanography, University of Rhode Island, Kingston, RI, personal communication, 1978. 14. Shaw, D. G., Institute of Marine Science, University of Alaska, College, AK, personal communication, 1977. 15. Calder, J. Α., Florida State University, personal communication, 1977. 16. LaFlamme, R. E.; Hites, R. A. "The Global Distribution of Polycyclic Aro matic Hydrocarbons in Recent Sediments," Geochim. Cosmochim. Acta 1978, 42, 289. 17. Youngblood, W. W.; Blumer, M. "Polycyclic Aromatic Hydrocarbons in the Environment: Homologous Series in Soils and Recent Marine Sedi ments," Geochim. Cosmochim. Acta 1975, 39, 1303. 18. Dixon, W. J. "Processing Data for Outliers," Biometrics 1953, 9, 74. ;
RECEIVED October 31, 1978.
