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Trace water-soluble components from Prudhoe bay crude oil: Chemical characterization and mutagenicity
Marine Environmental Research 14 (1984) 506 510
Trace Water-Soluble Components from Prudhoe Bay Crude Oil: Chemical Characterization and Mutagenicity
John M. Kennish* & Scott B. Frencht Chemistry Department, Universityof Alaska, 3221 ProvidenceDrive, Anchorage,Alaska 99508, USA
We here report on an investigation of the chemical composition of the water-soluble fraction (WSF) from Prudhoe Bay crude oil (PBCO), and on studies to correlate biological activity in the Ames S a l m o n e l l a microsome assay with the observed composition. Two liter samples of Prudhoe Bay crude oil water-soluble fraction (see Fig. 1) were extracted either with three 5 ml aliquots of hexane, or a 50 ml aliquot of methylene chloride followed by two 25 ml aliquots, in silanized separatory funnels. The organic phase was evaporated to dryness with nitrogen and the residue weighed. Alternatively, 20 ml of XAD-2 resin was added to 2 liters of the WSF and stirred for 24 h, after which the resin was Soxhlet extracted overnight with methylene chloride, the solvent evaporated with nitrogen purging, and the residue weighed. After weighing, small samples (10-50 mg) of the crude extract residues were diluted to 3 ml with hexane and then fractionated into seven fractions on an activated silicic acid column (Yu & Hites, 1981). Each fraction was evaporated to dryness with nitrogen, weighed, dissolved in 25-100 pl of 2:1 methylene chloride-methanol and analyzed on a Hewlett-Packard 5986A mass spectrometer system with direct capillary column interfacing (Bean et al., 1980). The identification of the G C - M S peaks was * Temporary address: Cooperative Institute for Research in Environmental Sciences (CIRES), Universityof Colorado, Boulder, Colorado 80309, USA. t Kristine C. Knudsen award winner for excellencein undergraduatechemistry 1982. Present address: Chemistry Department, Indiana University, Bloomington, Indiana 47405, USA. 5O6 Marine Environ. Res. 0141-1136/84/$03"00 (~ ElsevierApplied Science Publishers Ltd,
England, 1984. Printed in Great Britain.
Trace water-soluble eomponents Jrom Prudhoe bay crude oil i
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2 TIME
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1. Prudhoe Bay crude oil water-soluble fraction equilibration time at pH 8. Prudhoe Bay crude oil water-soluble fraction was generated in silanized Pyrex carboys containing distilled water adjusted to pH 8 with NaHCO 3. The bottles were sealed with cleaned aluminum foil-linedparafilm, and stirred with a Teflon bar at room temperature (ca. 22 °C) for 4 days, without formation of an emulsion. Equilibration time was found to be approximately2 days by measuring the UV absorbance at 260 nm, but the systemwas allowed to stir for 4 days to ensure that equilibrium was reached for all compounds. Followingequilibration, the systemwas allowed to stand undisturbed for 24 h before the aqueous phase was siphoned. Fig.
accomplished by using available standards, GC retention indices (Vassilaros et al., 1982), comparison of observed spectra with published spectra and fundamental interpretation. The quantity of low volatility organic material recovered from WSF was found to vary significantly with the nature of the extractant. Methylene chloride was substantially more effective at removing organic matter from the WSF than was either XAD-2 or hexane (2.37 _+ 0.69 mg extracted per liter (N = 3) as compared to 1.40 + 0.01 mg/liter (N = 2) and 0.25 _+ 0.04mg/liter ( N = 5) for MeC12, XAD and hexane, respectively). Similar results have been noted in a study where mutagenic material was extracted from sewage effluent (Grabow et al., 1981). Table 1 shows the percentage of each of the seven fractions of WSF from residues of approximately 10 50rag extracted by the various methods. The hexane fraction was found to contain normal hydrocarbons C~4 through C32 as well as the branched hydrocarbons pristane and phytane. The hexane toluene 1"1 fraction contained principally two- or three-ring PAHs ranging from naphthalene through alkylphenanthrenes, and
508
John M . Kennish, Scott B. French
TABLE 1 Percentage Composition of Prudhoe Bay Crude Oil Water-Soluble Fraction Subfractions Fraction
10 m g ~
28 m g b
35 m g c
50 m g ~
Hexane Hexane/toluene ( 1: 1) Toluene Methylene chloride Methylene chloride/methanol (2: 1) Methylene chloride/methanol (1:2) Methanol
7.5 18.5 9-0 8.7 41.0 8'2 7.5
0.3 17.3 3.0 7-0 68.5 3.6 0.3
1.8 7.4 3.0 5-0 80.5 2-1 0.3
7-7 15.3 8.3 9.5 57.5 1.0 0.5
Extracted with hexane, b Extracted with XAD-2. c Methylene chloride. These values must be viewed as a semi-quantitative measure of the class fractionation of the original extract (Warner et al., 1979).
oxygen- and sulfur-containing heterocycles including dibenzofurans and dibenzothiophenes. No molecular ions above m / z 220 were observed in this fraction. The toluene fraction contained mainly carbazole and alkylcarbazoles. The largest molecular ion observed was m / z 237. The methylene chloride fraction contained mainly phenols, some nitrogen heterocycles, and higher molecular weight polar compounds which remain unidentified. The methylene chloride-methanol 2:1 fraction contained phenols, like those in the methylene chloride fraction, and a large number of polar compounds with fragmentation patterns indicative of phenolic acids. The remaining fractions contained traces of these polar compounds. No ions above m/z 237 were observed in any fraction. The results indicated that XAD-2 and hexane extract the same general classes of compounds as methylene chloride, but are less efficient in removing the more polar classes, such as found in the methylene chloride-methanol 2:1 fraction. The wide range of chemical classes found in WSF during this study is not surprising considering the chemical composition of PBCO. It is interesting, however, that only two- and three-ring aromatic compounds were observed. These results are in general agreement with earlier studies (Roubal et al., 1978; Bean et al., 1980; Malins et al., 1980; Stuermer et al., 198 l) and might be expected from the relative solubilities of the different compound types in water. Following chemical characterization, each fraction was taken up in dimethylsulfoxide and tested for mutagenicity by the plate incorporation
Trace water-soluble components JJ'om Prudhoe bay crude oil
509
method as described by Ames et al. (1975). The Salmonella typhimurium tester strains TA100 and TA98, supplied by Ames, were used. The mutagenesis assays were performed in triplicate with and without rat liver S-9 (Litton Bionetics, Inc.). Sample sizes of 10 1000/~g/plate were tested for each fraction, excluding the hexane fraction (Pelroy & Petersen, 1981). All such tests were negative, in general agreement with similar studies conducted on PBCO and weathered PBCO (Warner et al., 1979: Guerin et al., 1981). These results may be due to the low concentration of four- and five-membered ring compounds in our extracts, although we did detect potentially mutagenic alkylphenanthrenes and alkylfluoranthenes (Yu & Hites, 1981). However, the relatively low concentration of these compounds with respect to the total sample composition, or the low solubility of the compounds in the aqueous bioassay medium (Warner et al., 1979), may have contributed to the negative result. This work was performed under Grant F-81-12 with the Alaska Council on Science and Technology. We wish to thank William Nelson for the gift of Prudhoe Bay crude oil, and the Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, for a visiting fellowship (J.M.K.).
REFERENCES Ames, R. N., McCann, J. & Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsomal mutagenicity test. Murat. Res. 31, 347 74. Bean, R. M., Blaylock, J. W. & Riley, R. C. (1980). Application of trace analytical techniques to a study of hydrocarbon composition upon dispersion of petroleum in a flowing seawater system. In: Petroleum in the marine environment (Petrakis, L. & Wiss, F.T. (Eds)). Advances in Chemistry Series No. 185, American Chemistry Society, 235 46. Grabow, W. O. K., Burger, J. S. & Holner, C. A. (1981). Comparison of liquid-liquid extraction and resin adsorption for concentrating mutagens in Ames Salmonella/microsome assay on water. Bull. Environ. Contam. Toxicol. 24, 442 9. Guerin, M. R., Rubin, I. B., Roa, T. K., Clark, B. R. & Epler, J. L. (1981). Distribution of mutagenic activity in petroleum and petroleum substitutes. Fuel 60, 282 8. Malins, D. C., Krahn, M. M., Brown, D. W., Macleod Jr, W. D. &Collier, T. K. (1980). The analysis of petroleum products in marine environments. Helgoland wiss. Meeresunters. 33, 257 71.
510
John M. Kennish, Scott B. French
Pelroy, R. A. & Petersen, M. R. (1981). Use of the Ames test in the evaluation of shale oil fraction. Environ. Health Perspect. 30, 191 -203. Roubal, W. T., Stranahan, S. I. & Malins, D.C. (1978). The accumulation of low molecular weight aromatic hydrocarbons of crude oil by coho salmon (Oncorhynchus kisutch) and starry flounder (Platichthys stellatus). Arch. Era, iron. Contam. Toxieol. 7, 237 49. Stuermer, D. H., Spies, R. B. & Davis, P. H. (1981). Toxicity of Santa Barbara seep oil to starfish embryos. I. Hydrocarbon composition of test solutions and field samples. Mar. Environ. Res. 5, 275 86. Vassilaros, D. L., Kong, R. C., Later, D, W. & Lee, M. J. (1982). Linear retention index system for polycyclic aromatic compounds. J. Chromatogr. 252, 1 20. Warner, J. S., Margard, W. L. & Anderson, J. W. (1979). Activity-directed fractionation of petroleum samples. Final Report, Battelle Columbus and Pacific Northwest Laboratories, Columbus, Ohio and Sequim, Washington, 21. Yu, M. L. & Hites, R. A. (1981). Identification of organic compounds on diesel engine soot. Anal. Chem. 53, 951 -4.
Trace Water-Soluble Components from Prudhoe Bay Crude Oil: Chemical Characterization and Mutagenicity
John M. Kennish* & Scott B. Frencht Chemistry Department, Universityof Alaska, 3221 ProvidenceDrive, Anchorage,Alaska 99508, USA
We here report on an investigation of the chemical composition of the water-soluble fraction (WSF) from Prudhoe Bay crude oil (PBCO), and on studies to correlate biological activity in the Ames S a l m o n e l l a microsome assay with the observed composition. Two liter samples of Prudhoe Bay crude oil water-soluble fraction (see Fig. 1) were extracted either with three 5 ml aliquots of hexane, or a 50 ml aliquot of methylene chloride followed by two 25 ml aliquots, in silanized separatory funnels. The organic phase was evaporated to dryness with nitrogen and the residue weighed. Alternatively, 20 ml of XAD-2 resin was added to 2 liters of the WSF and stirred for 24 h, after which the resin was Soxhlet extracted overnight with methylene chloride, the solvent evaporated with nitrogen purging, and the residue weighed. After weighing, small samples (10-50 mg) of the crude extract residues were diluted to 3 ml with hexane and then fractionated into seven fractions on an activated silicic acid column (Yu & Hites, 1981). Each fraction was evaporated to dryness with nitrogen, weighed, dissolved in 25-100 pl of 2:1 methylene chloride-methanol and analyzed on a Hewlett-Packard 5986A mass spectrometer system with direct capillary column interfacing (Bean et al., 1980). The identification of the G C - M S peaks was * Temporary address: Cooperative Institute for Research in Environmental Sciences (CIRES), Universityof Colorado, Boulder, Colorado 80309, USA. t Kristine C. Knudsen award winner for excellencein undergraduatechemistry 1982. Present address: Chemistry Department, Indiana University, Bloomington, Indiana 47405, USA. 5O6 Marine Environ. Res. 0141-1136/84/$03"00 (~ ElsevierApplied Science Publishers Ltd,
England, 1984. Printed in Great Britain.
Trace water-soluble eomponents Jrom Prudhoe bay crude oil i
'
I
'
I
I
'
I
'
! 5
i
507
007
~
0.06
0 005 •
f
Ld 0 0 4 (..) Z 0.03 r~ 0 U3 0.02 m 0.01
u
230
•
250
275 X,NM
, 0
I I
,
I
i
2 TIME
I 3
r
] 4
I
1 6
(days)
1. Prudhoe Bay crude oil water-soluble fraction equilibration time at pH 8. Prudhoe Bay crude oil water-soluble fraction was generated in silanized Pyrex carboys containing distilled water adjusted to pH 8 with NaHCO 3. The bottles were sealed with cleaned aluminum foil-linedparafilm, and stirred with a Teflon bar at room temperature (ca. 22 °C) for 4 days, without formation of an emulsion. Equilibration time was found to be approximately2 days by measuring the UV absorbance at 260 nm, but the systemwas allowed to stir for 4 days to ensure that equilibrium was reached for all compounds. Followingequilibration, the systemwas allowed to stand undisturbed for 24 h before the aqueous phase was siphoned. Fig.
accomplished by using available standards, GC retention indices (Vassilaros et al., 1982), comparison of observed spectra with published spectra and fundamental interpretation. The quantity of low volatility organic material recovered from WSF was found to vary significantly with the nature of the extractant. Methylene chloride was substantially more effective at removing organic matter from the WSF than was either XAD-2 or hexane (2.37 _+ 0.69 mg extracted per liter (N = 3) as compared to 1.40 + 0.01 mg/liter (N = 2) and 0.25 _+ 0.04mg/liter ( N = 5) for MeC12, XAD and hexane, respectively). Similar results have been noted in a study where mutagenic material was extracted from sewage effluent (Grabow et al., 1981). Table 1 shows the percentage of each of the seven fractions of WSF from residues of approximately 10 50rag extracted by the various methods. The hexane fraction was found to contain normal hydrocarbons C~4 through C32 as well as the branched hydrocarbons pristane and phytane. The hexane toluene 1"1 fraction contained principally two- or three-ring PAHs ranging from naphthalene through alkylphenanthrenes, and
508
John M . Kennish, Scott B. French
TABLE 1 Percentage Composition of Prudhoe Bay Crude Oil Water-Soluble Fraction Subfractions Fraction
10 m g ~
28 m g b
35 m g c
50 m g ~
Hexane Hexane/toluene ( 1: 1) Toluene Methylene chloride Methylene chloride/methanol (2: 1) Methylene chloride/methanol (1:2) Methanol
7.5 18.5 9-0 8.7 41.0 8'2 7.5
0.3 17.3 3.0 7-0 68.5 3.6 0.3
1.8 7.4 3.0 5-0 80.5 2-1 0.3
7-7 15.3 8.3 9.5 57.5 1.0 0.5
Extracted with hexane, b Extracted with XAD-2. c Methylene chloride. These values must be viewed as a semi-quantitative measure of the class fractionation of the original extract (Warner et al., 1979).
oxygen- and sulfur-containing heterocycles including dibenzofurans and dibenzothiophenes. No molecular ions above m / z 220 were observed in this fraction. The toluene fraction contained mainly carbazole and alkylcarbazoles. The largest molecular ion observed was m / z 237. The methylene chloride fraction contained mainly phenols, some nitrogen heterocycles, and higher molecular weight polar compounds which remain unidentified. The methylene chloride-methanol 2:1 fraction contained phenols, like those in the methylene chloride fraction, and a large number of polar compounds with fragmentation patterns indicative of phenolic acids. The remaining fractions contained traces of these polar compounds. No ions above m/z 237 were observed in any fraction. The results indicated that XAD-2 and hexane extract the same general classes of compounds as methylene chloride, but are less efficient in removing the more polar classes, such as found in the methylene chloride-methanol 2:1 fraction. The wide range of chemical classes found in WSF during this study is not surprising considering the chemical composition of PBCO. It is interesting, however, that only two- and three-ring aromatic compounds were observed. These results are in general agreement with earlier studies (Roubal et al., 1978; Bean et al., 1980; Malins et al., 1980; Stuermer et al., 198 l) and might be expected from the relative solubilities of the different compound types in water. Following chemical characterization, each fraction was taken up in dimethylsulfoxide and tested for mutagenicity by the plate incorporation
Trace water-soluble components JJ'om Prudhoe bay crude oil
509
method as described by Ames et al. (1975). The Salmonella typhimurium tester strains TA100 and TA98, supplied by Ames, were used. The mutagenesis assays were performed in triplicate with and without rat liver S-9 (Litton Bionetics, Inc.). Sample sizes of 10 1000/~g/plate were tested for each fraction, excluding the hexane fraction (Pelroy & Petersen, 1981). All such tests were negative, in general agreement with similar studies conducted on PBCO and weathered PBCO (Warner et al., 1979: Guerin et al., 1981). These results may be due to the low concentration of four- and five-membered ring compounds in our extracts, although we did detect potentially mutagenic alkylphenanthrenes and alkylfluoranthenes (Yu & Hites, 1981). However, the relatively low concentration of these compounds with respect to the total sample composition, or the low solubility of the compounds in the aqueous bioassay medium (Warner et al., 1979), may have contributed to the negative result. This work was performed under Grant F-81-12 with the Alaska Council on Science and Technology. We wish to thank William Nelson for the gift of Prudhoe Bay crude oil, and the Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, for a visiting fellowship (J.M.K.).
REFERENCES Ames, R. N., McCann, J. & Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsomal mutagenicity test. Murat. Res. 31, 347 74. Bean, R. M., Blaylock, J. W. & Riley, R. C. (1980). Application of trace analytical techniques to a study of hydrocarbon composition upon dispersion of petroleum in a flowing seawater system. In: Petroleum in the marine environment (Petrakis, L. & Wiss, F.T. (Eds)). Advances in Chemistry Series No. 185, American Chemistry Society, 235 46. Grabow, W. O. K., Burger, J. S. & Holner, C. A. (1981). Comparison of liquid-liquid extraction and resin adsorption for concentrating mutagens in Ames Salmonella/microsome assay on water. Bull. Environ. Contam. Toxicol. 24, 442 9. Guerin, M. R., Rubin, I. B., Roa, T. K., Clark, B. R. & Epler, J. L. (1981). Distribution of mutagenic activity in petroleum and petroleum substitutes. Fuel 60, 282 8. Malins, D. C., Krahn, M. M., Brown, D. W., Macleod Jr, W. D. &Collier, T. K. (1980). The analysis of petroleum products in marine environments. Helgoland wiss. Meeresunters. 33, 257 71.
510
John M. Kennish, Scott B. French
Pelroy, R. A. & Petersen, M. R. (1981). Use of the Ames test in the evaluation of shale oil fraction. Environ. Health Perspect. 30, 191 -203. Roubal, W. T., Stranahan, S. I. & Malins, D.C. (1978). The accumulation of low molecular weight aromatic hydrocarbons of crude oil by coho salmon (Oncorhynchus kisutch) and starry flounder (Platichthys stellatus). Arch. Era, iron. Contam. Toxieol. 7, 237 49. Stuermer, D. H., Spies, R. B. & Davis, P. H. (1981). Toxicity of Santa Barbara seep oil to starfish embryos. I. Hydrocarbon composition of test solutions and field samples. Mar. Environ. Res. 5, 275 86. Vassilaros, D. L., Kong, R. C., Later, D, W. & Lee, M. J. (1982). Linear retention index system for polycyclic aromatic compounds. J. Chromatogr. 252, 1 20. Warner, J. S., Margard, W. L. & Anderson, J. W. (1979). Activity-directed fractionation of petroleum samples. Final Report, Battelle Columbus and Pacific Northwest Laboratories, Columbus, Ohio and Sequim, Washington, 21. Yu, M. L. & Hites, R. A. (1981). Identification of organic compounds on diesel engine soot. Anal. Chem. 53, 951 -4.