13C12c ratios in individual fatty acids of marine mytilids with and without bacterial symbionts

Org. Geochem. Vol. 21. No. 6/7, pp. 611-617, 1994

~)

Pergamon

13C/12C ratios

0146-6380(93)E0021-D

Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights 0146-6380/94$7.00+ 0.00

reserved

in individual fatty acids of marine mytilids with and without bacterial symbionts

T. A. ABRAJANOJR, l D. E. MURPHY,l* J. FANG,2 P. COMET2 and J. M. BROOKS2 IDepartment of Earth Sciences, Memorial University of Newfoundland, St John's, Newfoundland, Canada AIB3X5 and 2Geochemical and Environmental Research Group, Texas A&M University, College Station, TX 77845, U.S.A.

Abstract--Noevidence was found for isotopic fractionation at methyl carbon during methylation of fatty

acids with BF3-methanol.It is therefore possible to evaluate intermolecular carbon isotopic variations in natural fatty acids by determining the 6 ~3C composition of the corresponding methyl esters after derivatization. We illustrate the usefulnessof the technique to the evaluation of dietary strategies of marine mytilids in two contrasting marine environments, a normal coastal Newfoundland estuarine system and a cold hydrocarbon seep benthic community from the Gulf of Mexico. Mussels from coastal Newfoundland have fatty acid 6 ~3C compositions of -34.4 to -24.9%o, whereas those from the Gulf of Mexico showed a range of -56.9 to -49.0%0. This difference is primarily ascribed to the differencein the ultimate source of CI carbon in the two environments (CO2 in coastal Newfoundland and CH4 in the Gulf of Mexico). Differences observed in the nature of intermolecular carbon isotopic patterns of saturated and unsaturated fatty acids of mytilids from the two marine localities reflect the differences in the carbon pathways utilized by the organisms in their respective growth environments. Key words---compound-specific carbon isotope analysis, mytilids, fatty acids, methyl esters, hydrocarbon

seep, estuary, bacterial symbionts

INTRODUCTION Compound-specific isotope analyses (CSIA) in complex organic mixtures will undoubtedly lead to very significant developments in geochemistry and other fields (Sano et al., 1976; Matthews and Hayes, 1978). The technique combines separation of individual compounds by gas-liquid chromatography, quantitative combustion of eluting compounds to CO2 in the combustion interface, and sequential isotopic analysis of CO2 by IRMS. Because water and potentially other gases are produced at the combustion interface, a mechanism for purifying CO2 before reaching the mass spectrometer was also incorporated in existing instrumental designs. It has also been shown that isotopic fractionation accompanies transport of individual compounds through the GC column (e.g. Hayes et al., 1990). Therefore, methods for proper integration of real time ion beam signals across individual peaks were also developed for GC/C/IRMS. Numerous discussions of instrument design, capabilities and limitations are now available (e.g. Barrie et al., 1984; Freedman et al., 1988; Hayes et al., 1990). Of the many notable considerations relevant to the proper application of the technique, procedures used *Present address: School of Earth and Ocean Sciences, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2.

for dealing with background, peak overlap and coelution deserve the most attention. When compounds are not fully resolved by the GC or high background signals underlie the peaks (especially if the background isotopic composition is very different from that of the peak), degradation of precision and accuracy can be expected. Hence careful sample preparation and separation prior to chromatography are required for more reproducible results. Although attempts have been made to analyze 2H/IH and 15N/14N in individual compounds, most published reports deal exclusively with 13C/12C determination. Isotopic results are reported in conventional delta (6) notation, where 6 13C~= 1000 (gs/RpD B - 1) (R represents the ratio 13Cp2C and the subscripts s and PDB refer to sample and standard Pee Dee Belemnite, respectively). In general, quoted precision and accuracy are 0.3%o or better, although these often only consider instrumental precision for very well resolved sample peaks. In this paper, we examine a procedure for determining the carbon isotopic compositions of individual fatty acids. Attention has been focused on the range of fatty acids found in marine mytilids (see below). Of particular analytical concern has been the potential for a kinetic isotope effect (KIE) during fatty acid esterification. Esterification is a necessary step for chromatographic separation and, hence, for the GC/C/IRMS analysis of individual fatty acids. Because this step results in the addition of carbon to

611

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T . A . A e ~ A n o JR et al.

the free fatty acid, it is imperative that we develop an understanding of how to derive the carbon isotope composition of the free fatty acid from the measured carbon isotopic composition of the ester. If no significant fractionation of isotopes occurs during derivatization, a mass balance relation can be used to estimate the isotopic composition of the free fatty acids. Even if fractionation does occur during derivatization, it may be possible to correct for it if the fractionation is experimentally reproducible (e.g. Silfer et al., 1991). To demonstrate the potential of the technique in unravelling carbon pathways in marine ecosystems, we also present preliminary results of carbon isotopic analyses of fatty acids extracted from mussels from two distinct growth environments. One group of mussels was recovered from shallow estuaries around Newfoundland and the other from a deep, cold (as opposed to hydrothermal) hydrocarbon-seep environment from the Gulf of Mexico. Previous bulk carbon isotopic investigations of mytilid mussels from the Gulf of Mexico have revealed extreme depletions of ~3C, implicating endosymbiotic methanotrophic bacteria as an important contributor to the mussels diet (e.g. Kennicutt et al., 1992). This in turn implies the utilization of biogenic or thermogenic methane as the primary carbon substrate for the benthic community around the gas seeps. In contrast, similar carbon isotopic studies of marine mytilids from normal estuarine environments showed less depleted x3C values for the mussel tissues, although slightly depleted ~3C values have been encountered in some reducing estuarine environments such as sewage outfalls and mangrove swamps (Felbeck and Somero, 1982). The isotopic composition of individual fatty acids is controlled by the nature and availability of the carbon source and the isotopic fractionation accompanying metabolism and biosynthesis in the animals. Our goal was to examine whether these factors can be distinguished in the fatty acid isotopic signature of marine mytilids. The results confirm the value of CSIA of fatty acids in defining food sources and biochemical pathways in mussels. These compound-specific isotopic data should complement previous evaluation of carbon flow in benthic ecosystems on the basis of fatty acid molecular characterization. METHODS

Pure fatty acid standards and BFa-methanol were acquired from Supelco, Inc. (Bellefonte, Pa). Fatty acid methyl esters (FAME) were formed by reaction with BF3-methanol at 100°C. When reaction with BF3-methanol was complete, the esters were extracted into dichloromethane and the solvent was evaporated. An aliquot of the product was weighed for conventional isotopic measurement, and the remainder taken up in dichloromethane for GC injection. Replicate samples (i.e. standard fatty acids and

esters), approx. 1-3 mg each, were loaded into precleaned pyrex tubes (6 mm o.d. sealed at one end). Excess pre-combusted CuO was added to each tube, which was subsequently evacuated and flame sealed. The evacuated samples were combusted in a 550°C furnace overnight (cf. Sofer, 1980). The CO2 produced was purified cryogenically and the amount measured manometricaUy. The CO2 gas was collected into pre-evacuated sample holders and immediately analyzed by IRMS. Extreme precautions were followed during sample collection and storage. The procedures followed for sample collection and storage for the Gulf of Mexico mytilids have been described previously (Fang et aL, 1993a, 1993b). For the shallow Newfoundland estuaries, mussels were retrieved by divers and kept frozen until ready for use. Frozen mussel tissues were freeze-dried, weighed and saponified with 6 N KOH. The non-saponifiable fraction was separated by repeated extraction with methylene chloride and stored in a freezer for future separation and analysis. The basic aqueous solution was acidified with concentrated HC1 to pH 1 and the fatty acids were separated by repeated extraction with methylene chloride. The separated fatty acids were then esterified using BF3-methanol. Aliquots of solutions of free fatty acids and methyl esters were evaporated to dryness and prepared for bulk isotopic analyses as described above for pure standards. Another aliquot of the fatty acid methyl esters was chromatographically characterized using procedures described previously (Fang et al., 1993a). The carbon isotopic compositions of the total free fatty acids, the corresponding mixture of methyl esters, and the BF3-methanol were determined by conventional IRMS. These bulk isotopic measurements were performed using VG Prism and Finnigan MAT 252 isotope ratio mass spectrometers. Cross calibration of samples and standards was performed on both instruments to ensure consistency of results. Compound-specific G C / C / I R M S analyses of fatty acid methyl esters were performed using a VG Isochrom system equipped with a Hewlett-Packard 5890 gas chromatograph [30m x 0.25ram SPB-5 (Supelco) and DB-5 (J&W Scientific) columns, 20 psi column head pressure]. Standards and samples were taken up in hexane before injection. For all the G C / C / I R M S analyses, standardization was accomplished by comparing integrated 13Cp2C for each compound peak with similar ratios from pulses of reference CO2 gas introduced before and after the sample chromatographic window. The accuracy of this procedure was tested by repeated analyses of our fatty acid carbon isotopic standards. However, we also spiked several of our samples with internal isotopic standards to allay concerns over the reliability of the standardization and background correction procedures. The values measured for the internal isotopic standards are all well within the

13C/12C ratios in individual fatty acids

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Table I. Carbon isotopiccompositionsof fatty acid standardsand derivedesters* 6 '3C (~o) Methyl ester Methyl ester Methylester Fatty acid Fatty a c i d conventional GC/C/IRMS predicted A~" 10:0

ND

-28.50(0.19)

-29.02(0.18)

ND

ND

12:0 ND -31.75(0.08) -31.54(0.16) ND ND 14:0 -26.13(0.12) -27.32(0.04) -26.98(0.21) -27.47 -0.15 16:0 -26.73 (0.03) -27.93(0.12) -27.57(0.12) -28.11 -0.18 18:0 -29.46(0.19) -30.59(s) -30.51 (0.20) -30.34 +0.25 20:0 -25.13(0.07) -26.28(s) -26.10(0.07) -26.43 -0.16 22:0 -26.96(0.12) -28.18(0.12) -28.38(0.20) -27.97 +0.21 24:0 -28.26(0.09) -28.55(0.10) -28.78(0.15) -29.17 -0.62 *Numbers in parentheses represent Io deviation from the mean. Predicted methyl ester isotopic compositions are those derived from equation (I) (see text). For equation (I), a 6 13C value of -50.10%ewas usedfor the BFrmethanol(determinedby conventionalIRMS).ND, not determined; (s), singleanalysisonly. l'Differencebetweenmeasuredand predicted methylester 6 ~3C. analytical precision reported here. In general, the dilution factor was such that a 1 #1 injection of sample or standard contained 10 nmol of CO2 from the most abundant methyl ester. With a split injection of about 30:1 and an open split of 20:1, the most abundant compound in a 1/~1 injection yielded a major ion beam (i.e. mass 44) signal of around 10 - s A. Quantitation and identification of individual fatty acids was accomplished through the use of external standards (see Fang et al., 1993a).

RESULTS AND DISCUSSION Precision and accuracy

Instrumental precision* with our conventional isotope ratio mass spectrometers is better than 0.01%o. The 6 13C value of our secondary reference gas for the GC/C/IRMS was calibrated against NBS-16, NBS-17 and NBS-19. Using our adopted value for the secondary reference CO2 gas, the maximum deviations of our measured isotopic values and the accepted values for the three primary standards are: NBS-16 (-0.14%o). NBS-17 (+0.05%o) and NBS-19 (+0.04%o). The measurement accuracy attributable to our reference CO 2 should therefore be around 0.1%o. Replicate bulk carbon isotope analysis of pure fatty acid standards taken through the saponification and esterification steps differ by < 0.2%o. An experimental accuracy of better than 0.3%0 for the bulk carbon isotopic measurements was determined from a comparison between fatty acid standards that were combusted directly and those taken through the saponification and extraction steps prior to combustion. The limiting accuracy and precision for GC/C/IRMS analysis are primarily dictated by the (1) amount of compound injected, (2) extent of coelution with other compounds, and (3) back-

ground. For both the standards and samples, the background is very clean and the correction required is minimal (as confirmed by use of internal isotopic standards). To ensure that the ion-beam signals for each peak analyzed were within the linear dynamic range of the mass spectrometer (i.e. 10-9-10 -8 A), several sample injections were performed for each sample. The multiple-injection precision routinely achieved for well resolved peaks was better than 0.2%O, whereas the worst precision accepted for poorly resolved doublets was 0.5%o [i.e. peaks that cannot be replicated to within a value of 0.5%o (la deviation) are excluded from the data analysis]. The latter is largely dependent on the isotopic contrast between the chromatographically overlapping compounds. An accuracy of better than 0.4%o for CSIA was deduced from a comparison between the 6 13CFAMEvalues measured by GC/C/IRMS and conventional IRMS (Table 1, Fig. 1). However, we caution that the standards we used are all saturated fatty acids, and the corresponding methyl esters are chromatographically very well resolved. The column background is also very low in all cases examined in this work. Our experience indicates that where background levels are such that background corrections

-26

e/•

C10:0

C~/~

ZCo -32

I

*In this paper, precisions and reproducibilities are reported as l~r deviation from the mean of the measured parameter. Unless otherwise stated, the accuracy values quoted are maximum deviations from the accepted values.

C24:0

L

I

I

613(3 (Conventional IRMS)

Fig. 1. Comparison of ~ a3C values measured by GC/C/ IRMS and conventional IRMS for esterified fatty acid standards.

T.A. AeRAJ^NO JR et al.

614

Carbon isotope abundances in fatty acids of marine mytilids

~°E, l "--,o ]

/ C a ~ n Number

Fig. 2. Difference observed between measured ~ 13C and predicted 6 ~3C plotted against carbon number. Bracketing lines represent deviations of 0.3%0.

are required, i n t r o d u c t i o n o f several well positioned isotopic s t a n d a r d s is imperative.

Kinetic isotope effect during derivatization T h e f o r m a t i o n o f fatty acid methyl ester from the free fatty acid involves the addition of one m e t h a n o l c a r b o n per fatty acid molecule. If the c a r b o n from m e t h a n o l a d d e d to the fatty acid molecule is isotopically identical to the p a r e n t m e t h a n o l molecule, a mass balance relation of the form:

6 13CFAME-~- [X ]6 13CFA"[- (1 -- x)6 13CcH3oH

(1)

can be used to estimate the c a r b o n isotope c o m p o s i t i o n o f the free fatty acid (6 13CFg from the k n o w n isotopic c o m p o s i t i o n o f m e t h a n o l c a r b o n (613CcH3OH) a n d the m e a s u r e d isotopic c o m p o s i t i o n of the methyl ester (6 13CFAME). In e q u a t i o n (1), x is the fractional c a r b o n c o n t r i b u t i o n o f the free fatty acid to the ester. F o r example, x has the value of 16/17 a n d 20/21 for the esterification of hexadecanoic acid and eicosanoic acid, respectively. Table 1 (also see Fig. 2) c o m p a r e s the 6 13C values predicted by e q u a t i o n (1) to those measured by GC/C/IRMS and conventional I R M S . With the possible exception o f the tetracosanoic acid, the predicted a n d m e a s u r e d values agreed to within the precision limits of the G C / C / I R M S measurements. Also note t h a t the calculated isotopic difference between the predicted a n d measured values can be positive or negative. To be consistent with a kinetic isotope effect at the methyl c a r b o n during derivatization, the m e a s u r e d 6 ~3C values should have been consistently lower t h a n the predicted values. These results do not imply t h a t n o kinetic isotope effect occurs at the carboxyl position during esterification. The m o r e a p p r o p r i a t e conclusion is t h a t n o isotopic fractionation o f consequence to e q u a t i o n (1) was f o u n d u n d e r the conditions used in o u r experiments.

In order to evaluate the m a g n i t u d e o f variations t h a t m a y exist in the c a r b o n isotopic compositions of individual fatty acids, we examined two sets of n a t u r a l fatty acid samples. F a t t y acids were extracted from m a r i n e mytilids from shallow estuarine systems a r o u n d N e w f o u n d l a n d a n d the results are c o m p a r e d to those reported from the deeper waters o f the G u l f o f Mexico ( F a n g et al., 1993b). T h e mussels examined from b o t h localities belong to the family Mytilidae [Mytilus edulis a n d Modiolus modiolus for the N e w f o u n d l a n d mussels a n d seep mytilid Ib for the G u l f o f Mexico mussels ( F a n g et al., 1993a)]. Two sets o f mussel samples were collected a r o u n d the A v a l o n Peninsula of N e w f o u n d land: (1) Bellevue Beach, Trinity Bay, a n d (2) Holyrood, C o n c e p t i o n Bay. The seep mussels from the G u l f o f Mexico are from a n A l a m i n o s C a n y o n dive site located at 2 6 ° 2 1 ' N a n d 94°30"W ( F a n g et al., 1993a). These seep mussels have previously been s h o w n to contain m e t h a n o t r o p h i c bacterial symbionts (e.g. F a n g et al., 1993a, b; K e n n i c u t t et al., 1992).

Table 2. Representative fatty acid compositions of marine mytilids from Newfoundland and Gulf of Mexico Concentrations (#g/g) Peak Newfoundland Gulf of Mexico No .Fatty acid* (NFH-14) (W18260) I 13:0 2209.1 -14:1n7 -68.4 2 3 14:0 2343.0 236.7 4 15:1 -40.2 15:0 2454.0 lOI.4 5 16:1n9 -2924.9 6 7 16:ln7 4104.3 1657.2 8 16:0 12332.5 2013.9 17:1n14 -19.8 9 I0 17:0 -37.2 II 18:4n3 1199.6 -12 18:3n7 -1678.8 18:2n10 -274.5 13 14 18:2n7 -323.7 15 18:1n9 2854.2 402.0 18:1n7 -1098.9 16 18:0 1590.3 501.6 17 18 19:2t -36.3 19 19:1n8 -108.0 20:5n3 5643.0 -20 21 20:3t -170.1 22 20:3n9 -1156.5 23 20:2n9 -1293.6 20:2n8 -103.8 24 25 20:1n9 1855.6 733.5 26 20:1n7 -1556.7 20:0 1069.2 -27 21:5n3 439.8 -28 29 22:6n3 2459.0 -30 22:5n3 597.2 -22:3n7 -63.6 31 32 22:2n12 -104.1 33 22:2n7 -728.1 34 24:4t -128.3 24:1t 392.7 -35 *The symbol 20:5n3 denotes a 20 carbon long fatty acid with 5 double bonds, and where the first double bond from the methyl end is between the third and fourth carbon.

tPositions of double bonds unknown.

13C/t2C ratios in individual fatty acids JSF-03 •

3

8 7

15 11

17

NR<-14

et al. (1993) reported that 6 13C values of gaseous

N_~..

hydrocarbons issuing from these cold seep settings ranged from - 8 0 to -35%0. Some of the fatty acids reported in Fig. 3 for the Gulf of Mexico mussels may have derived directly from bacteria (not necessarily symbionts), as opposed to mussel tissues. Whereas the extent of unsaturation observed in the fatty acids is not normally expected for bacterial fatty acids (e.g. Perry et al., 1979; Bobbie and White, 1980), relatively little is known about fatty acid distribution in seep bacterial symbionts, and it is not possible to distinguish specific, direct contributions from bacteria to the measured fatty acid pool (e.g. Fang et al., 1993a). Nevertheless, we have shown previously that no significant differences exist between fatty acid distributions in the gill and foot tissues of mussel samples from the Gulf of Mexico (Fang et al., 1993b). This indicates that bacteria that coat the gills of symbiontcontaining mussels are only minor contributors to the fatty acid inventory of the mussels analyzed. Also notable are the patterns of intermolecular t513C variation in fatty acids from single organism. Large intra-organism 6 ]3C variations are noted in mussels from both localities: 9%o for the Newfoundland mussels and 7%o for the Gulf of Mexico mussels. The exact origin of these variations cannot be fully ascertained at present, although they probably reflect the combined effects of (1) isotopically distinct dietary sources for specific fatty acids and (2) isotopic variations accompanying de novo fatty acid synthesis or modification (Fang et al., 1993a, b; Monson and Hayes, 1982). It is possible, for example, that isotopic fractionation accompanying chain elongation and desaturation could be imparting distinct isotopic signatures to the fatty acids (Fang et al., 1993b). Factors controlling these suggested biosynthetic fractionations have been examined previously by Monson and Hayes (1982). In both localities, depletion in~3C appears to accompany increasing degree of desaturation. The greatest depletions of ]3C were found in fatty acids with the highest degree of unsaturation. The depletions of 13C in monounsaturated relative to saturated fatty acids average about 2%0. This relationship, if substantiated (preferably experimentally) and refined by subsequent measurements, could be useful in distinguishing isotopic variations due to de novo biosynthesis effects from dietary (mixing) effects. For example, the extremely depleted value for 18:4n3 compared to 18:0 in the Newfoundland mussels, could indicate additional "unique" input to the mussel tissue of this specific compound. This is consistent with the less extreme depletion of t3C noted in 20:5n3 compared to 20:0. Indeed, the 6 ~3C composition of 20:5n3 overlaps with those of 22:6n3, and the fact that these compounds are both significantly heavier than 18:4n3 makes it unlikely that the latter two compounds originated by de novo elongation of the ]3C-depleted 18:4n3. The KIE expected for chain elongation from 18: 4n3 to 20: 5n3 and 22: 6n3 should

20 28 25

29

35 30

NF

.............

-30

E .as 8

0

6

-50

16 12

23

34

17

26

I

I

GM

455 6

615

I

:

12

Carbon Number

Fig. 3. Comparison of compound-specific GC/C/IRMS 6 t3C analyses of fatty acids. GM, Gulf of Mexico (samples labelled JSF); NF, Newfoundland estuaries (samples labelled NFB [Bellevue] and NFH [Holyrood], see text). Each analysis represents CSIA determination on fatty acids extracted from homogenized whole bodies of one organism. Peak numbers correspond to those identified in Table 2.

Abundances of individual fatty acids for representative samples from the two localities are shown in Table 2 (also see Fang et al., 1993a, b for detailed discussion). Figure 3 compares the isotopic compositions (8 1 3 C F A ) of individual fatty acids from the two populations (see Fig. 4 for a sample trace of m / z 44 from the GC/C/IRMS ). In both cases, the 6 J3C values reported are those of the underivatized fatty acids [equation (1)]. The isotopic values shown in Fig. 3 are consistent with the bulk fatty acid compositions (Newfoundland: -27.3 + 0.7%0 (mean + la); Gulf of Mexico: --51.6 + 2.2%0) and the bulk tissue compositions (Newfoundland: - 2 4 . 0 + 0.3%0; Gulf of Mexico: - 4 8 . 6 + 0.5%0) measured for both localities. The contrast between the 6 '3CFA of the two mussel populations reflects the fundamentally different sources of carbon for fatty acid synthesis in the two environments. The Newfoundland mussels thrived in a normal marine setting in which phytoplankton provided the ultimate food source. The range of 6 ~3C values measured in this case is lower than the bulk ~ 13C of the phytoplankton from the same locality [ca. - 2 7 to -22%0 (Ostrom, 1992)]. This difference is to be expected because the 6 ~3C of the bulk organic carbon is always higher by several per rail compared to the fatty acid fraction (e.g. Parker, 1964; DeNiro and Epstein, 1977). In contrast, the carbon isotopic composition of fatty acid extracts from the Gulf of Mexico hydrocarbon seep mussels reflect little, if any, contribution from phytoplankton. Instead, the ultimate carbon source for fatty acid synthesis appears to have been methane issuing from seeps around which the mussels and other benthic organisms flourish (Fang et al., 1993a, b). Kennicutt

616

T.A. ABRAJANOJR et al.

have made the latter two lighter, not heavier, than 18:4n3 (Fang et ai., 1993b; Monson and Hayes, 1982). In the case of the Gulf of Mexico mussels, we also previously reported (Fang et al., 1993b) what appears to be a systematic depletion of ~3C in the longer fatty acids with a similar degree of unsaturation. This effect, ascribed to fractionation accompanying chain elongation (Fang et al., 1993), was not observed in the Newfoundland mussels. In the Newfoundland mussels, for example, the series 16: 0, 18 : 0 and 20: 0 are isotopically overlapping within individual tissue samples. The reason for this is unclear, although we speculate that the difference may be related to the differing carbon pathways in the two environments of mussel growth. Mussels from the Gulf of Mexico grew under very "restrictive" conditions, whereby they have to depend on a single chemosynthetic process to form the carbon substrate for fatty acid biosynthesis. These mussels had to rely almost exclusively on endosymbiotic methanotrophic bacteria that in turn relied on methane as a carbon source.

1.1=-8

The carbon pathway of fatty acid biosynthesis utilized by the seep organisms was therefore minimized enabling maximum expression of a kinetic isotope effect. The isotopic signature of the biosynthcsized fatty acid in the normal environment of growth of Newfoundland mussels, in contrast, is characterized by a multiplicity of carbon sources and pathways. The Newfoundland mussels grew in an environment with a larger diversity of food sources. This situation enables mixed dietary intake to dominate the isotopic signatures of the resultant fatty acids. It is conceivable that particular dietary sources will dominate the contribution to specific fatty acid chain lengths or even specific compounds (e.g. 18:4:n3) in the mussel tissue (e.g. Paradis and Ackman, 1977; Ackman, 1989). The fact that systematic 13C depletions in the polyunsaturates are nevertheless observed in Newfoundland mussels implies that isotopic fractionations accompany chain desaturation reactions are much larger than those associated with chain elongation and the variations imposed by dietary diversity (although the desaturation may well have

Newfoundland Mussel (FA-14)

20

1 .E-8

5.E-9 0


ij,7

29

15

4.E-9

28

11

t/} u)

3.E-9

2.E-9

~'.

30

]

T°°

3

~

35

""

1 .E-9

0.E+0

27 IIIIIII1[1111

85O

Illlllllllllllilllllllllllllllllllllll 1000

1150

1300

14S0

Retention time (sec)

Fig. 4. Chromatographic trace of m/z = 44 for fatty acids from a Newfoundland mussel in the GC/C/ IRMS. Peak numbers correspond to those identified in Table 2.

13C/12C ratios in individual fatty acids occurred in the food source). We speculate that in ecologically well defined systems, CSIA measurements could offer a means of quantitatively assessing the magnitude of specific dietary input to specific organisms (of. Paradis and Ackman, 1977). CONCLUSION Much will be learned as more mussel occurrences are studied by G C / C / I R M S . To further constrain specific speculations on dietary intake, it is imperative that a more comprehensive compound-specific isotope investigation of larger groups of compounds from entire ecosystems is conducted. It may also be instructive, after establishing normal isotopic and dietary patterns in unperturbed environments, to compare these patterns to ecosystems affected by various types of h u m a n activities. It is possible that perturbation in carbon flow caused by increased pollution will leave distinctive marks on the compound-specific carbon isotope signatures of fatty acids or other compounds in mussels and other marine organisms. Acknowledgements--The U.S. Department of Energy Office of Basic Energy Sciences provided funding for the GC/C/IRMS facility and initial exploratory research on fatty acids at Argonne National Laboratory/University of Chicago. Memorial University's new GC/C/IRMS facility was funded by the Natural Sciences and Engineering Research Council (Canada). B. Holt, N. Sturchio, P. Lobel, C. Parrish, M. Wilson, V. O'MaUey, and P. Eakin provided valuable assistance at various stages of the project. The comments of J. Hayes and three anonymous reviewers greatly clarified the interpretation and presentation of the present results. REFERENCES

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