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Comparison of sampling and extraction techniques for fatty acids in recent sediments
735
Notes Oeochimica et Cosmochimics Acta, 1971,Vol. 35. pp. 735 to 741. Pergamon Press. Printedin Northern Ireland
Comparison of sampling and extraction techniques for fatty acids in recent sediments JOHN
W.
FARRINOTON
Graduate School of Oceanography,
University
(Received 18 November 1970;
and JAMES
G.
QUINN
of Rhode Island, Kingston,
accepted in revisedform
13 March
Rhode Island 02881 1971)
Abstract-Saponification of recent, sediment samples was the most efficient of several procedures tested for the extraction of fatty acids. Between 32 and 65 per cent of the fatty acids in the sediments analyzed could not be released by organic solvent extraction. This fraction was released by saponification and is thought to be bound fatty acid. The precision of the analysis is an improvement over those previously reported for concentrations of fatty acids in sediments. Relative and absolute abundances of fatty acids from sediments give valid criteria for comparison of depositional environments only when uniform sampling and extraction techniques are employed.
INTRODUCTION THE CROWING awareness of sediment pollution problems necessitates formulation of valid criteria for comparing recent sediments from polluted and unpolluted Comparison of different depositional environments depositional environments. using organic compounds as criteria, as suggested by PARKER (1969) when reviewing the work of PETERSON (1967) on fatty acids in recent sediments, would seem to require consistent sampling and extraction techniques. Conversely, the application of different extraction techniques to sediment samples might release specific fractions of a particular class of organic compounds thereby yielding information about the interaction of these compounds with other organic matter and minerals in the sediments being studied. Fatty acid analyses have been reported for a wide variety of sediments. These analyses have been reviewed by KVENVOLDEN (1967, 1970), PETERSON (1967), PARKER (1969), and DOUGLAS et al. (1970). Organic solvent extraction enhanced by heating, Soxhlet apparatus, and sonication; saponification (COOPER, 1962; KVENVOLDEN, 1966); and partial demineralization using HF: HCl prior to solvent extraction (VAN HOEVEN et al., 1969) are all commonly used to extract fatty acids. PARKER (1969) has noted that there has been no systematic comparison of different extraction methods by different workers on a standard sample. Unfortunately, we do not have data of this kind to report. However, we have conducted a series of experiments applying several extraction techniques to recent sediments, and have related these to the efficiency for one technique, thereby giving a common basis for comparison. EXPERIMENTAL Sampling The general sedimentary environment of Narragansett Bay has been discussed by MCMASTER (1960). Samples were obtained using a Foster Anchor Dredge which selects approximately the upper 8 cm of sediment supplemented by 50 cm Phleger cores. Three dredge hauls were taken at each station and subsamples of each dredge haul were analyzed either separately or combined.
736
Notes
All subsamples were frozen and stored at -20°C upon arrival in the laboratory two to six hours after sampling. Samples were wet sieved using distilled water through a 1 mm screen to remove any larger organisms, rocks and shells before extraction. A check of the washings showed negligible losses of fatty acid. Moisture content of the sediment sample was determined by drying duplicate (l-5 g) sediment samples at 1OO’C to constant weight. Two hours was found to be sufficient. Extractions All chemicals used in this study were A.C.S. reagent grade. The solvents were distilled in an all glass still fitted with a 30 cm Widmer column. Sediment samples were all 20-50 g dry-weight and contained 25-50% moisture when extracted. Crs:o* (nonadecanoic acid) was added as an internal standard to the sediment samples prior to extraction.? 1. Saponi’cation extraction. This procedure is a modification of that used by COOPER (1962) and KVENVOLDEN (1966). The sediment sample was refluxed for two hours in a 500 ml round bottom flask with 200 ml of 0.5 N KOH in 95 % methanol. The sediment was allowed to settle and the solvent decanted through a Buchner funnel containing Whatman No. 1 filter paper into a 1 1. filtering flask. The sediment in the round bottom flask was stirred for 5 minutes on a magnetic stirrer with 200 ml of (1: 1) methanol: 1.3 N hydrochloric acid. This mixture was filtered through the same paper into the same filter flask, and the sediment washed on the filter paper with 200 ml of (1: 1) methanol : water followed by 200 ml of petroleum ether. Tho combined filtrate and washings (pH Q 2) were transferred to a 1 1. separatory funnel and vigorously shaken. The petroleum ether phase was isolated and washed with 100 ml of The ether phase was then drawn through anhydrous sodium sulfate which had been water. prewashed with distilled chloroform, and the solvent evaporated under reduced pressure. The procedure for analysis of the residue obtained after solvent evaporation is described below. Replacement of the 2 hr reflux with blending for 15 min was tested, as was replacement of the 200 ml of 0.5 N KOH in 95% methanol with 200 ml of 0.5 N KOH in 95% methanol: benzene (1:l). 2. So&let e&action. Whatman cellulose single thickness 43 x 123 mm extraction thimbles were extracted for 72 hr with the solvent mixture to be used for the sediment extraction. Sediment samples were extracted for 48 hr. 3. Sonication. A Bronwill Biosonik Probe was used to extract the sediment samples according to the procedure of PARKER and LEO, (1965). 4. Direct solvent rejihx. Sediment samples were refluxed for 2 hr in the solvent. The procedure was used as described for biological 5. BLICH and DYER (1959) extraction. samples with the exception of doubling the blending times. The lipid extracts from 2 to 5 were saponified, acidified, and re-extracted with petroleum under reduced pressure. The sediment residues ether. The petroleum ether was evaporated of l-3 were re-extracted using the saponification procedure of 1 with the exception that Cso;J (eicosanoic acid) was added as an internal standard to the sediment residues prior to extraction. The lipid extracts from petroleum ether evaporation for procedures l-5 were transferred to screw cap centrifuge tubes with teflon lined caps using 1 ml of methanol and 1 ml of benzene. Two ml of BFs-methanol were added and the extract heated at 100°C for five minutes. The methyl esters were isolated and purified by preparative thin layer chromatography. Isolation of fatty acids prior to methylation was not necessary as a check showed no interference with the methylation by other compounds in the extract. The methyl esters were analyzed on F & M Model 1609 and Hewlett Packard Model 700 gas chromatographs equipped with flame ionization detectors. The columns used were a * Fatty acids are designated chain 1ength:number t Crs:~ occurs naturally in trace amounts. $ Cso:o occurred naturally in trace amounts.
of double bonds.
Notes
737
3 mm o,d. x 2-I m 15% stabilized DEGS on Gas Chrom P-80/100 mesh and a 3 mm o.d. x 18 m 10% UCCW-982 (Hewlett Packard-silicone gum rubber) on Chromosorb W-SO/IO0 mesh. Columns were routinely operated isothermally at 180% and 200% respectively with & nitrogen Comparison of relative retention times with those of carrier gas flow rate of 60-74 ml/mm standard methyl esters before and after hydrogenation served to tentatively identify the acids. The areas of naturally occurring component peaks were compared to the area of the internal standard peak to obtain quantitative results. The accuracy of the gas chromatographic analyses, determined by the method of HERB and MARTIN (19’70), is indicated by a value of 96.51 per cent using NIH mixture 1). The maximum coefficient of variation observed for any one fatty acid in this mixture was 2.7 1 per cent as determined from four analyses. Blanks for the extraction procedures were less than 1 per cent of the lowest concentration of fatty acids found in the sediment.
The sum of concentrations of the fatty acids extracted by several procedures (1-3) and the fatty acids extracted by saponification of the sediment residue from the initial extraction agreed with the concentrations of fatty acids extracted by saponification alone. The coefficient of variation for the saponification procedure, including natural variability among subsamples of dredge hauls and cores at the same station, ranged from 2 to 42 per cent and averaged at 18 per cent, based upon five sets of triplicate analysis with six major fatty acids per set. The reproducibility of the analyses of subsamples from three dredge hauls w&s the same as three analyses of a mixture of the same subsamples. This is a great improvement on the reproducibility of orders of magnitude reported by PETERSON (1967)who used a sonication technique. proA cheek on the absolute recovery of the Crs:e internal standard for the saponification cedure was 46-57 per cent. Low concentr%tio~ of the unsaturated fatty acids of chain length C,, and above were found and might have resulted from preferential degradation of these acids during the extraction. To check on this, duplicates of a standard mixture of fatty acids C&e, C2*:I, C&:8 and Western Bentonite clay were extracted using the %:l~ %:lz c20:1, C22:13 c20:5* saponification procedure. There was little destruction of the polyuns&urated fatty acids which suggests that the fatty acids extracted from sediments using the saponification procedure are representative of the in situ distribution. Preferential destruction of unsaturated fatty acids may occur during some extraction procedures. We tested direct methylation extraction of the fatty acids involving refluxing sediment samples previously washed with methanol to remove the water in 2 and 20% I&SO, in methanol. Analysis of the fatty acids and methyl esters extracted by both the methanol wash and extraction respectively showed that there were severe losses of the C&r and C&r fatty acids, probably from oxidlttion. Substitution of 200 ml of 1: 1 benzene: 0.5 N KOH in 95 % methanol did not decrease the efficiency of the saponification extraction for fatty acids, and probably increased the efficiency for extracting hydrocarbons. KVENOLDEW (1966) noted that one drawback to his sapo~fication procedure was the low solubility of hydrocarbons in methanol. Blending for 15 min in place of a 2 hr reflux gave the same results for the s~ponific&tion extraction. However, this was set aside due to the danger of using a non-explosion proof blendor. The relative abundance of fatty acids compared to the Cro:e internal standard extracted by the second saponification was the same as the relative abundance of the fatty acids extracted by the first saponification. Our interpretation is that fatty acids in the second extract resulted from adsorption of the fatty acids in the first extract onto mineral particles during filtering, and remained with the sediment residue. This is supported by the fact that at most the additional fatty acids extracted by the second saponification amounted to only 5 per cent of those extracted by the first saponification. The concentrations of fatty acids extracted from dredge samples of recent sediments using various extraction techniques, and saponification of core samples are listed in Table 1. The six major fatty acids which comprised 75-80 per cent of the tot& fatty acids detected by our analyses in the range Cre:o-C&~ were used in this study to compare sampling and extraction techniques. Table 1 shows that saponification yields greater amounts of fatty acids than organic solvent extraction. The organic solvent extractions applied to the same sediment sample
X&B
738
(Station X8---Table 1) seem Lcryield fairly consistent results which is probably due to their extracting the same fraction of fatty acids, that is the unbound fatty acids, These acids are herein de&gm&ed as iaeids and esters easily extra&abIe by orgaGe solvents c.g, free fatty acids ~tnd waxes. Bffm~d f&y acids are those CtGidsand esters extracted by sa~o~~~~t~o~ of the sediment samples after organic3 solvent 0xtraction e.g. (1) fatty acids (MEYERS and @.~NN, 1971) iand their esters associated with clays and/or incorporated into humic materials (OUNE:~ and SNITZER, 1970a, b) (2) fatty acids esterified to OH groups of humia molecules. The d&a of Tables f arzd 2 &arty ~~rn~~~~~~~ of different dep~~~~o~~ envkonments OX the basis of fatty acid eonoentrations or relativs abundance is only valid when consistent sampling and extraction procedures have been employed. Comparison of core samples with dredge samples can be biased by the derrroasing conosntrations of fatty acids and deereasing relative ablmdance of C~s:l and C1szl fatty a&da with increasing depth ix1 shart ~~~faee cores O-50 cm ~~~R~~~~~~ and @mm, 1971; Pazam and LEO, 1965). Dependirrg on the rate of diagenesis, the cantz-ibutions of the lower sections of thlo core may be overshadowed by the higher concentrations in the surface 8oction as could be the ease for Station E, (Table 2), or manifest themselves in alter@ the relative abundance of fatty acids for the whole core as could be the case fop St&ion C (Table 2). Fin&y, ret&&-e abundance of fatty a&% in the u%bound state which pw’e extracted by organic solvents alons, and the bound fatty acids which are extracted by saponification after
149 f 5-J f
9.0
4.7
4.5
27.9
If-1
4.7
3.6
26-7
6-3 7.9
3-4 5-O
2.6 2.9
19-2 24.5
14.7
9.7
7.3
45.0
28.8 & 4.4 20.2
5-1
4.9 1.4
8.3
IS?
104 + 2.3 7.6
7.2 f 1.3 1.9
6.3
3-6
IO-9
5.1
50.6
10.7
4.0
34.5
739
I-Totes
organic solvent extraction differ as shown in Table 2. There is a consistently higher relative abundance of Crezl and Crs:r in the unbound fatty acid fraction extracted by two different Since the total amounts organic solvent systems, and organic solvents enhanced by sonication. of fatty acids extracted by organic solvent techniques followed by saponification is the s&me as the amounts extracted by saponification alone, the lower relative abundance of unsaturated fatty acids cannot be attributed to degradation during successive extractions. Table
Station
2. Comparison
of abundances
of fatty
acids relative
to Crs:c (palmitic
acid)*
C1,t:o
Cl&t-
cl63
%3:0
%3:1
0.61 0.56
0.60 0.70
0.67 0.59
0.24 0.26
0.40 0.32
0.33 0.66 0.28 0.42
0.41 0.35 0.35 0.20
0.52 0.39 0.66 o-41
0.33 0.19 0.27 0.12
0.50 0.25 0.49 0.26
O-56 0.51
0.45 0.40
o-57 0.38
0.18 0.15
0.25 0.09
0.45 0.55
0.61 0.41
0.78 O-51
0.30 0.19
043 0.23
0052
0.50
0.69
o-19
0.33
0.44 0.53
0.57 0.47
2.2 0.60
0.19 0.19
0.44 0.25
E,
S~ponifieation Dredge Core Solvent
Extraction
Soxhlet-ChC&:
(Dredge)
MeOH-
Sonic&ion-CHCls-MeOH Station
A:. B A B
C
Saponification Dredge Core Solvent
Extraction
(Dredge)
Soxhlet-CHCl,:MeOH Station
B (Dredge
A I3
only)
Saponification _____ Solvent
Extrrtction
Soxhlet-benzene:
MeOH
A B
* Values calculated from data in Table 1 with exception of B-bound fatty acids. t Mixture of unresolved C,, (n, &so, urzteiso) and C& fatty acids. $ A extracted by solvent extraction-~bo~lnd fatty acids. B extracted by saponification of sediment residue from solvent extraction-bound acids.
fatty
DISCUSSION The extraction of bound fatty acids by saponification could be due to (1) the K+ ion alteration of the affinity of mineral surfaces towards fatty acids or the formation of the K+ salt of the fatty acid promoting greater solubility thereby decreasing adsorption of the fatty acid onto the mineral surface, (2) cleaving ester linkages to humic materials if such linkages occur, (3) solubilization of humic materials releasing entrapped fatty acids. OONER and SNITZER (lQ70a, b) suggest that fatty acids are entrapped within fulvic acid, a humie material. Between 32 and 65 per cent of the fatty acids in the recent sediments we investigated were bound fatty acids. DOUGLAS et al. (1970) have shown that the total 7
Notes
740
fatty acids released by saponification of an Eocene sample of Green River shale were ten times that of the fatty acids released by sonication with benzene and methanol. Although much more work is needed, especially on samples of an intermediate geological age, it appears that bound fatty acids are stabilized relative to unbound fatty acids over geological time or diagenesis of fatty acids and other organic compounds produces bound fatty acids. An understanding of the biogeochemistry of many classes of organic compounds in recent sediments is necessary in order to define the seriousness and longevity of sediment pollution by specific organic compounds. This investigation demonstrates the need for employing uniform sampling and extraction techniques when comparing different sedimentary environments, e.g. clean and polluted, on the basis of the qualitative and quantitative distribution of organic compounds. CONCLUSIONS (1) Saponification extracts a greater amount of fatty acids from recent sediments than sonication, Soxhlet, or organic solvents alone. (2) Comparisons of sedimentary environments on the basis of fatty acid distribution could be misleading if different extraction and sampling techniques are used. (3) Between 32 and 65 per cent of the fat’ty acids in recent sediments exist in a bound condition. Acknozc;ledgements-Supported by Pre-Doctoral Fellowship So. 5-Fl-WP-26,426.02 from the Federal 1Vater Quality Administration (J. W. F.) and National Science Foundation Sea Grant No. GH-99. We thank Dr. DONALD PHELPS and his staff (National Marine Water Quality Laboratory, West Kingston, Rhode Island) for their assistance in collecting the sediment samples. REFERENCES BLICH E. G. and DYER W. T. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. COOPERJ. E. (1962) Fatty acids in recent and ancient sediments and petroleum reservoir waters. Nature
193,744-746.
DOUGLASA. C., DOURAGHI-ZADEHK., EGLINTONG., MAXWELLJ. R. and RAMSEY J. N. (1970) Fatty acids in sediments including the Green River Shale (Eocene) and Scottish Torbanite (Carboniferous). In Advances in Organic Geochemistry, 1966 (editors G. D. Hobson and G. C. Speers). Pergamon Press. FARRINGTONJ. W. and QUINN J. G. (1971) Fatty acid diagenesis in recent sediments from Narragansett Bay, Rhode Island. Nature Phys. Sci. 230, 67-69. HERB S. F. and MARTINV. G. (1970) How good are analyses of oils by GLC. J. Amer. Oil Chem. Sot. 47, 415-421.
KVENVOLDENK. A. (1970) Evidence for transformations of normal fatty acids in sediments. In Advances in Organic Geochemistry, 1966 (editors G. D. Hobson and G. C. Speers). Pergamon Press. KVENVOLDENK. A. (1967) Normal fatty acids in sediments. J. Amer. Oil Chem. Sot. 44, 628-636.
KVENVOLDENK. A. (1966) Molecular distribution of normal fatty acids and paraffins in some lower Cretaceous sediments. Nature 209,573-577. MCMASTER R. L. (1960) Sediments of Narragansett Bay System and Rhode Island Sound, Rhode Island. J. Sediment Petrol. 30, 249-274.
Notes
741
MEYERS P. A. and QUINN J. G. (1971) Fatty acid-clay mineral associations in artificial and natural sea water solutions. Geochim. Cosmochim. Acta 35, 628-632. OGNER G. and SNITZER M. (1970a) The occurrence of alkanes in fulvic acid, a soil humic fraction. Beochim. Cosmochim. Acta 34, 921-928. phythalate complexes OGNER G. and SNITZER M. (1970b) Humic substances: fulvic acid-dialkyl and their role in pollution. Science 170, 317-318. In Organic Geochemistry (editors G. Eglinton PARKER P. L. (1969) Fatty acids and alcohols. and M. T. J. Murphy). Springer. PARKER I’. L. and LEO R. F. (1965) Fatty acids in Blue Green Algal Mat Communities. SC~PVLCA 148, 373-374. PETERSON U. H. (1967) Fatty acid composition of certain shallow water marine sediment,s. Ph.D. Thesis, University of Washington. P.~N HOEVEN W., MAXWELL J. R. and CALVIN M. (1969) Fatty acids and hydrocarbons as evidence of life processes in ancient sediments and crude oils. Geochim. Cosmochim. ~&da 33, 877-881.
Notes Oeochimica et Cosmochimics Acta, 1971,Vol. 35. pp. 735 to 741. Pergamon Press. Printedin Northern Ireland
Comparison of sampling and extraction techniques for fatty acids in recent sediments JOHN
W.
FARRINOTON
Graduate School of Oceanography,
University
(Received 18 November 1970;
and JAMES
G.
QUINN
of Rhode Island, Kingston,
accepted in revisedform
13 March
Rhode Island 02881 1971)
Abstract-Saponification of recent, sediment samples was the most efficient of several procedures tested for the extraction of fatty acids. Between 32 and 65 per cent of the fatty acids in the sediments analyzed could not be released by organic solvent extraction. This fraction was released by saponification and is thought to be bound fatty acid. The precision of the analysis is an improvement over those previously reported for concentrations of fatty acids in sediments. Relative and absolute abundances of fatty acids from sediments give valid criteria for comparison of depositional environments only when uniform sampling and extraction techniques are employed.
INTRODUCTION THE CROWING awareness of sediment pollution problems necessitates formulation of valid criteria for comparing recent sediments from polluted and unpolluted Comparison of different depositional environments depositional environments. using organic compounds as criteria, as suggested by PARKER (1969) when reviewing the work of PETERSON (1967) on fatty acids in recent sediments, would seem to require consistent sampling and extraction techniques. Conversely, the application of different extraction techniques to sediment samples might release specific fractions of a particular class of organic compounds thereby yielding information about the interaction of these compounds with other organic matter and minerals in the sediments being studied. Fatty acid analyses have been reported for a wide variety of sediments. These analyses have been reviewed by KVENVOLDEN (1967, 1970), PETERSON (1967), PARKER (1969), and DOUGLAS et al. (1970). Organic solvent extraction enhanced by heating, Soxhlet apparatus, and sonication; saponification (COOPER, 1962; KVENVOLDEN, 1966); and partial demineralization using HF: HCl prior to solvent extraction (VAN HOEVEN et al., 1969) are all commonly used to extract fatty acids. PARKER (1969) has noted that there has been no systematic comparison of different extraction methods by different workers on a standard sample. Unfortunately, we do not have data of this kind to report. However, we have conducted a series of experiments applying several extraction techniques to recent sediments, and have related these to the efficiency for one technique, thereby giving a common basis for comparison. EXPERIMENTAL Sampling The general sedimentary environment of Narragansett Bay has been discussed by MCMASTER (1960). Samples were obtained using a Foster Anchor Dredge which selects approximately the upper 8 cm of sediment supplemented by 50 cm Phleger cores. Three dredge hauls were taken at each station and subsamples of each dredge haul were analyzed either separately or combined.
736
Notes
All subsamples were frozen and stored at -20°C upon arrival in the laboratory two to six hours after sampling. Samples were wet sieved using distilled water through a 1 mm screen to remove any larger organisms, rocks and shells before extraction. A check of the washings showed negligible losses of fatty acid. Moisture content of the sediment sample was determined by drying duplicate (l-5 g) sediment samples at 1OO’C to constant weight. Two hours was found to be sufficient. Extractions All chemicals used in this study were A.C.S. reagent grade. The solvents were distilled in an all glass still fitted with a 30 cm Widmer column. Sediment samples were all 20-50 g dry-weight and contained 25-50% moisture when extracted. Crs:o* (nonadecanoic acid) was added as an internal standard to the sediment samples prior to extraction.? 1. Saponi’cation extraction. This procedure is a modification of that used by COOPER (1962) and KVENVOLDEN (1966). The sediment sample was refluxed for two hours in a 500 ml round bottom flask with 200 ml of 0.5 N KOH in 95 % methanol. The sediment was allowed to settle and the solvent decanted through a Buchner funnel containing Whatman No. 1 filter paper into a 1 1. filtering flask. The sediment in the round bottom flask was stirred for 5 minutes on a magnetic stirrer with 200 ml of (1: 1) methanol: 1.3 N hydrochloric acid. This mixture was filtered through the same paper into the same filter flask, and the sediment washed on the filter paper with 200 ml of (1: 1) methanol : water followed by 200 ml of petroleum ether. Tho combined filtrate and washings (pH Q 2) were transferred to a 1 1. separatory funnel and vigorously shaken. The petroleum ether phase was isolated and washed with 100 ml of The ether phase was then drawn through anhydrous sodium sulfate which had been water. prewashed with distilled chloroform, and the solvent evaporated under reduced pressure. The procedure for analysis of the residue obtained after solvent evaporation is described below. Replacement of the 2 hr reflux with blending for 15 min was tested, as was replacement of the 200 ml of 0.5 N KOH in 95% methanol with 200 ml of 0.5 N KOH in 95% methanol: benzene (1:l). 2. So&let e&action. Whatman cellulose single thickness 43 x 123 mm extraction thimbles were extracted for 72 hr with the solvent mixture to be used for the sediment extraction. Sediment samples were extracted for 48 hr. 3. Sonication. A Bronwill Biosonik Probe was used to extract the sediment samples according to the procedure of PARKER and LEO, (1965). 4. Direct solvent rejihx. Sediment samples were refluxed for 2 hr in the solvent. The procedure was used as described for biological 5. BLICH and DYER (1959) extraction. samples with the exception of doubling the blending times. The lipid extracts from 2 to 5 were saponified, acidified, and re-extracted with petroleum under reduced pressure. The sediment residues ether. The petroleum ether was evaporated of l-3 were re-extracted using the saponification procedure of 1 with the exception that Cso;J (eicosanoic acid) was added as an internal standard to the sediment residues prior to extraction. The lipid extracts from petroleum ether evaporation for procedures l-5 were transferred to screw cap centrifuge tubes with teflon lined caps using 1 ml of methanol and 1 ml of benzene. Two ml of BFs-methanol were added and the extract heated at 100°C for five minutes. The methyl esters were isolated and purified by preparative thin layer chromatography. Isolation of fatty acids prior to methylation was not necessary as a check showed no interference with the methylation by other compounds in the extract. The methyl esters were analyzed on F & M Model 1609 and Hewlett Packard Model 700 gas chromatographs equipped with flame ionization detectors. The columns used were a * Fatty acids are designated chain 1ength:number t Crs:~ occurs naturally in trace amounts. $ Cso:o occurred naturally in trace amounts.
of double bonds.
Notes
737
3 mm o,d. x 2-I m 15% stabilized DEGS on Gas Chrom P-80/100 mesh and a 3 mm o.d. x 18 m 10% UCCW-982 (Hewlett Packard-silicone gum rubber) on Chromosorb W-SO/IO0 mesh. Columns were routinely operated isothermally at 180% and 200% respectively with & nitrogen Comparison of relative retention times with those of carrier gas flow rate of 60-74 ml/mm standard methyl esters before and after hydrogenation served to tentatively identify the acids. The areas of naturally occurring component peaks were compared to the area of the internal standard peak to obtain quantitative results. The accuracy of the gas chromatographic analyses, determined by the method of HERB and MARTIN (19’70), is indicated by a value of 96.51 per cent using NIH mixture 1). The maximum coefficient of variation observed for any one fatty acid in this mixture was 2.7 1 per cent as determined from four analyses. Blanks for the extraction procedures were less than 1 per cent of the lowest concentration of fatty acids found in the sediment.
The sum of concentrations of the fatty acids extracted by several procedures (1-3) and the fatty acids extracted by saponification of the sediment residue from the initial extraction agreed with the concentrations of fatty acids extracted by saponification alone. The coefficient of variation for the saponification procedure, including natural variability among subsamples of dredge hauls and cores at the same station, ranged from 2 to 42 per cent and averaged at 18 per cent, based upon five sets of triplicate analysis with six major fatty acids per set. The reproducibility of the analyses of subsamples from three dredge hauls w&s the same as three analyses of a mixture of the same subsamples. This is a great improvement on the reproducibility of orders of magnitude reported by PETERSON (1967)who used a sonication technique. proA cheek on the absolute recovery of the Crs:e internal standard for the saponification cedure was 46-57 per cent. Low concentr%tio~ of the unsaturated fatty acids of chain length C,, and above were found and might have resulted from preferential degradation of these acids during the extraction. To check on this, duplicates of a standard mixture of fatty acids C&e, C2*:I, C&:8 and Western Bentonite clay were extracted using the %:l~ %:lz c20:1, C22:13 c20:5* saponification procedure. There was little destruction of the polyuns&urated fatty acids which suggests that the fatty acids extracted from sediments using the saponification procedure are representative of the in situ distribution. Preferential destruction of unsaturated fatty acids may occur during some extraction procedures. We tested direct methylation extraction of the fatty acids involving refluxing sediment samples previously washed with methanol to remove the water in 2 and 20% I&SO, in methanol. Analysis of the fatty acids and methyl esters extracted by both the methanol wash and extraction respectively showed that there were severe losses of the C&r and C&r fatty acids, probably from oxidlttion. Substitution of 200 ml of 1: 1 benzene: 0.5 N KOH in 95 % methanol did not decrease the efficiency of the saponification extraction for fatty acids, and probably increased the efficiency for extracting hydrocarbons. KVENOLDEW (1966) noted that one drawback to his sapo~fication procedure was the low solubility of hydrocarbons in methanol. Blending for 15 min in place of a 2 hr reflux gave the same results for the s~ponific&tion extraction. However, this was set aside due to the danger of using a non-explosion proof blendor. The relative abundance of fatty acids compared to the Cro:e internal standard extracted by the second saponification was the same as the relative abundance of the fatty acids extracted by the first saponification. Our interpretation is that fatty acids in the second extract resulted from adsorption of the fatty acids in the first extract onto mineral particles during filtering, and remained with the sediment residue. This is supported by the fact that at most the additional fatty acids extracted by the second saponification amounted to only 5 per cent of those extracted by the first saponification. The concentrations of fatty acids extracted from dredge samples of recent sediments using various extraction techniques, and saponification of core samples are listed in Table 1. The six major fatty acids which comprised 75-80 per cent of the tot& fatty acids detected by our analyses in the range Cre:o-C&~ were used in this study to compare sampling and extraction techniques. Table 1 shows that saponification yields greater amounts of fatty acids than organic solvent extraction. The organic solvent extractions applied to the same sediment sample
X&B
738
(Station X8---Table 1) seem Lcryield fairly consistent results which is probably due to their extracting the same fraction of fatty acids, that is the unbound fatty acids, These acids are herein de&gm&ed as iaeids and esters easily extra&abIe by orgaGe solvents c.g, free fatty acids ~tnd waxes. Bffm~d f&y acids are those CtGidsand esters extracted by sa~o~~~~t~o~ of the sediment samples after organic3 solvent 0xtraction e.g. (1) fatty acids (MEYERS and @.~NN, 1971) iand their esters associated with clays and/or incorporated into humic materials (OUNE:~ and SNITZER, 1970a, b) (2) fatty acids esterified to OH groups of humia molecules. The d&a of Tables f arzd 2 &arty ~~rn~~~~~~~ of different dep~~~~o~~ envkonments OX the basis of fatty acid eonoentrations or relativs abundance is only valid when consistent sampling and extraction procedures have been employed. Comparison of core samples with dredge samples can be biased by the derrroasing conosntrations of fatty acids and deereasing relative ablmdance of C~s:l and C1szl fatty a&da with increasing depth ix1 shart ~~~faee cores O-50 cm ~~~R~~~~~~ and @mm, 1971; Pazam and LEO, 1965). Dependirrg on the rate of diagenesis, the cantz-ibutions of the lower sections of thlo core may be overshadowed by the higher concentrations in the surface 8oction as could be the ease for Station E, (Table 2), or manifest themselves in alter@ the relative abundance of fatty acids for the whole core as could be the case fop St&ion C (Table 2). Fin&y, ret&&-e abundance of fatty a&% in the u%bound state which pw’e extracted by organic solvents alons, and the bound fatty acids which are extracted by saponification after
149 f 5-J f
9.0
4.7
4.5
27.9
If-1
4.7
3.6
26-7
6-3 7.9
3-4 5-O
2.6 2.9
19-2 24.5
14.7
9.7
7.3
45.0
28.8 & 4.4 20.2
5-1
4.9 1.4
8.3
IS?
104 + 2.3 7.6
7.2 f 1.3 1.9
6.3
3-6
IO-9
5.1
50.6
10.7
4.0
34.5
739
I-Totes
organic solvent extraction differ as shown in Table 2. There is a consistently higher relative abundance of Crezl and Crs:r in the unbound fatty acid fraction extracted by two different Since the total amounts organic solvent systems, and organic solvents enhanced by sonication. of fatty acids extracted by organic solvent techniques followed by saponification is the s&me as the amounts extracted by saponification alone, the lower relative abundance of unsaturated fatty acids cannot be attributed to degradation during successive extractions. Table
Station
2. Comparison
of abundances
of fatty
acids relative
to Crs:c (palmitic
acid)*
C1,t:o
Cl&t-
cl63
%3:0
%3:1
0.61 0.56
0.60 0.70
0.67 0.59
0.24 0.26
0.40 0.32
0.33 0.66 0.28 0.42
0.41 0.35 0.35 0.20
0.52 0.39 0.66 o-41
0.33 0.19 0.27 0.12
0.50 0.25 0.49 0.26
O-56 0.51
0.45 0.40
o-57 0.38
0.18 0.15
0.25 0.09
0.45 0.55
0.61 0.41
0.78 O-51
0.30 0.19
043 0.23
0052
0.50
0.69
o-19
0.33
0.44 0.53
0.57 0.47
2.2 0.60
0.19 0.19
0.44 0.25
E,
S~ponifieation Dredge Core Solvent
Extraction
Soxhlet-ChC&:
(Dredge)
MeOH-
Sonic&ion-CHCls-MeOH Station
A:. B A B
C
Saponification Dredge Core Solvent
Extraction
(Dredge)
Soxhlet-CHCl,:MeOH Station
B (Dredge
A I3
only)
Saponification _____ Solvent
Extrrtction
Soxhlet-benzene:
MeOH
A B
* Values calculated from data in Table 1 with exception of B-bound fatty acids. t Mixture of unresolved C,, (n, &so, urzteiso) and C& fatty acids. $ A extracted by solvent extraction-~bo~lnd fatty acids. B extracted by saponification of sediment residue from solvent extraction-bound acids.
fatty
DISCUSSION The extraction of bound fatty acids by saponification could be due to (1) the K+ ion alteration of the affinity of mineral surfaces towards fatty acids or the formation of the K+ salt of the fatty acid promoting greater solubility thereby decreasing adsorption of the fatty acid onto the mineral surface, (2) cleaving ester linkages to humic materials if such linkages occur, (3) solubilization of humic materials releasing entrapped fatty acids. OONER and SNITZER (lQ70a, b) suggest that fatty acids are entrapped within fulvic acid, a humie material. Between 32 and 65 per cent of the fatty acids in the recent sediments we investigated were bound fatty acids. DOUGLAS et al. (1970) have shown that the total 7
Notes
740
fatty acids released by saponification of an Eocene sample of Green River shale were ten times that of the fatty acids released by sonication with benzene and methanol. Although much more work is needed, especially on samples of an intermediate geological age, it appears that bound fatty acids are stabilized relative to unbound fatty acids over geological time or diagenesis of fatty acids and other organic compounds produces bound fatty acids. An understanding of the biogeochemistry of many classes of organic compounds in recent sediments is necessary in order to define the seriousness and longevity of sediment pollution by specific organic compounds. This investigation demonstrates the need for employing uniform sampling and extraction techniques when comparing different sedimentary environments, e.g. clean and polluted, on the basis of the qualitative and quantitative distribution of organic compounds. CONCLUSIONS (1) Saponification extracts a greater amount of fatty acids from recent sediments than sonication, Soxhlet, or organic solvents alone. (2) Comparisons of sedimentary environments on the basis of fatty acid distribution could be misleading if different extraction and sampling techniques are used. (3) Between 32 and 65 per cent of the fat’ty acids in recent sediments exist in a bound condition. Acknozc;ledgements-Supported by Pre-Doctoral Fellowship So. 5-Fl-WP-26,426.02 from the Federal 1Vater Quality Administration (J. W. F.) and National Science Foundation Sea Grant No. GH-99. We thank Dr. DONALD PHELPS and his staff (National Marine Water Quality Laboratory, West Kingston, Rhode Island) for their assistance in collecting the sediment samples. REFERENCES BLICH E. G. and DYER W. T. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. COOPERJ. E. (1962) Fatty acids in recent and ancient sediments and petroleum reservoir waters. Nature
193,744-746.
DOUGLASA. C., DOURAGHI-ZADEHK., EGLINTONG., MAXWELLJ. R. and RAMSEY J. N. (1970) Fatty acids in sediments including the Green River Shale (Eocene) and Scottish Torbanite (Carboniferous). In Advances in Organic Geochemistry, 1966 (editors G. D. Hobson and G. C. Speers). Pergamon Press. FARRINGTONJ. W. and QUINN J. G. (1971) Fatty acid diagenesis in recent sediments from Narragansett Bay, Rhode Island. Nature Phys. Sci. 230, 67-69. HERB S. F. and MARTINV. G. (1970) How good are analyses of oils by GLC. J. Amer. Oil Chem. Sot. 47, 415-421.
KVENVOLDENK. A. (1970) Evidence for transformations of normal fatty acids in sediments. In Advances in Organic Geochemistry, 1966 (editors G. D. Hobson and G. C. Speers). Pergamon Press. KVENVOLDENK. A. (1967) Normal fatty acids in sediments. J. Amer. Oil Chem. Sot. 44, 628-636.
KVENVOLDENK. A. (1966) Molecular distribution of normal fatty acids and paraffins in some lower Cretaceous sediments. Nature 209,573-577. MCMASTER R. L. (1960) Sediments of Narragansett Bay System and Rhode Island Sound, Rhode Island. J. Sediment Petrol. 30, 249-274.
Notes
741
MEYERS P. A. and QUINN J. G. (1971) Fatty acid-clay mineral associations in artificial and natural sea water solutions. Geochim. Cosmochim. Acta 35, 628-632. OGNER G. and SNITZER M. (1970a) The occurrence of alkanes in fulvic acid, a soil humic fraction. Beochim. Cosmochim. Acta 34, 921-928. phythalate complexes OGNER G. and SNITZER M. (1970b) Humic substances: fulvic acid-dialkyl and their role in pollution. Science 170, 317-318. In Organic Geochemistry (editors G. Eglinton PARKER P. L. (1969) Fatty acids and alcohols. and M. T. J. Murphy). Springer. PARKER I’. L. and LEO R. F. (1965) Fatty acids in Blue Green Algal Mat Communities. SC~PVLCA 148, 373-374. PETERSON U. H. (1967) Fatty acid composition of certain shallow water marine sediment,s. Ph.D. Thesis, University of Washington. P.~N HOEVEN W., MAXWELL J. R. and CALVIN M. (1969) Fatty acids and hydrocarbons as evidence of life processes in ancient sediments and crude oils. Geochim. Cosmochim. ~&da 33, 877-881.