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Aliphatic hydrocarbons in particulate matter from the Baltic Sea
Marine Chemistry, 11 (1982) 55--70
55
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
A LI P HATI C HY D R O C A R B O N S IN P A R T I C U L A T E M A T T E R FROM T H E BALTIC SEA CHR. OSTERROHT and G. PETRICK Institut fiir Meereskunde an der Uniuersit;it Kiel, Diisternbrooker Weg 20, D-2300 Kiel (G.F.R.)
(Received March 20, 1981; revision accepted September 24, 1981) ABSTRACT Osterroht, Chr. and Petrick, G., 1982. Aliphatic hydrocarbons in particulate matter from the Baltic Sea. Mar. Chem., 11: 55--70. During May 1980, particulate matter was collected at an anchor station from different depths by two methods. In the separated hydrocarbon fraction the most abundant component was the highly unsaturated hydrocarbon n-heneicosahexaene (HEH). Also present was a series of mono- and di-unsaturated olefinic hydrocarbons with an uneven number of carbon atoms, starting with n-ClT. The alkane pattern was characterized by a maximum in the range between n-pentadeeane and n-heptadecane, with a strong uneven to even carbon number predominance. The peculiar composition of the aliphatic hydrocarbons was compared with literature data given for aliphatic hydrocarbons, whether produced by different marine organisms or derived from crude oil of other anthropogenic sources. A suggestion is made for using a set of conditions as indicators for pollution by fossil hydrocarbons. INTRODUCTION It is generally agreed t h a t particles having a diameter of m ore than 0.45 #m m ay be considered as particulate m a t t e r and all material with a smaller diameter is said to be dissolved, either colloidally or in molecular dispersion. Particulate m a t t e r m a y be divided into t w o categories: living organisms and detrital material (Cauwet, 1978). Most planktonic organisms are mainly f o u n d in the euphoric layer which in the Baltic Sea extends to 20 m dept h on average and is subject to a rapid and t h o r o u g h turnover. Detritus, however, is very stable with respect to minerals, remainders of living organisms and some organic com pounds , be i t adsorbed or incorporated. Therefore, the c o n c e n t r a t i o n of particulate organic carbon in the euphoric zone is n o t only higher by a factor of 5--10 relative to concentrations at greater dept h (Wangersky, 1974, 1976), but t he pat t er n of organic molecules in surface water particulate m a t t e r also reflects the state o f the water b o d y in question, i.e. biological activity as well as atmospheric input, land run off, etc. The relatively stable h y d r o c a r b o n s associated with suspended material, especially, are capable of reflecting the state of the environment, as shown by m a n y authors. F r o m gas chromatographic h y d r o c a r b o n patterns o f particles collected in sediment.traps (Crisp et al., 1979) and from filtered particulate material (Morris et al., 1977), biosynthetic origin as well as pollution, seepage or
0304-4203/82/0000-0000/$02.75 01982 Elsevier Scientific Publishing Company
56 other chemogenic origins can be inferred. In addition, several authors have demonstrated that particulate matter is a vehicle for the transport of hydrocarbons and renders the underlying sediments an indicator of processes in the water column (Lytle and Lytle, 1979). In tank experiments it was shown that aliphatics of a crude oil were predominantly associated with particulate matter and deposited on sediments, leading to a gas chromatographic pattern from the sediment, typical of the aliphatics of the crude oil. It was also confirmed that the extent of adsorption is inversely proportional to the solubility of the compounds in water, which means that the more soluble aromatics were depleted on suspended matter. Partition concentrations of the crude oil to water, particulate matter and sediments were the same as those found in the Baltic Sea after the Tsesis oil spill (Gearing et al., 1980; Wade and Quinn, 1980). On the other hand, polycyclic aromatic hydrocarbons (PAH), if sufficiently concentrated in the water, are also transported by suspended material to the sediments, as shown by Prahl and Carpenter (1979) by the remarkable similarity of PAH patterns in plankton, trap material and sediments. The Baltic Sea is a land-locked sea characterized by high river inputs of terrestrial material and a typical b o t t o m topography, characterized by three basins, separated by sills. The Gotland Deep (the area of this investigation) is the largest and deepest of these basins. Nevertheless, the distribution of particulate matter in the Gotland Deep follows the previously mentioned pattern of Wangersky: particulate organic carbon is most highly concentrated in the surface water, followed by a maximum of atomic ratio N/Cpart" at a b o u t 10 m, indicating that primary production is most intensive in this zone (Ehrhardt, 1969). Data of simultaneously investigated plankton patterns support this conclusion (Derenbach, 1969). Total concentrations of hydrocarbons, dissolved in water and adsorbed onto particulate material in the Gotland Deep, were measured by Zsolnay (1971) with a CHN-analyzer as hydrocarbon-derived organic carbon. The aim of this work was to evaluate the content of hydrocarbons on particulate matter and to compare the reresults with other data. Materials and methods
During May 1980, at an anchor station in the Gotland Sea (see Fig. 1 for geographic localities), samples were taken either by an automatic sampling b u o y (6 samples) from 1 m depth or from a depth of approximately 5 m by a ship-borne pumping system permitting pH control of the sampled water (12 samples). For technical details of sampling devices see Ehrhardt (1978) and Kremling et al. (1981). Material collected on filters by both sampling procedures was used for this investigation. All solvents were analytical grade, redistilled in an all-glass still over a 1 5 0 c m column filled with glass spirals; water was purified with a Milli-Q system by Millipore, including mixed-bed exchanger, activated charcoal and
57
Fig. 1. Map of the area of investigation. 0.2 tzm filter. Filters were Schleicher & Schull glass-fibre filters No. 6, heated in a drying cabinet at 300°C in an oxygen atmosphere. After filtration (for one sample about 4001 of seawater in the buoy system and about 2001 of seawater in the pH-controlled procedure) the filters were homogenized with diethylether by means of a Cenco high-speed homogenizer, resulting in a first extraction of lipophilic organic material under mild thermal conditions. After phase separation, the ether was decanted and the remaining homogenate was suspended in water free of organic carbon. The aqueous phase was extracted three times with an additional portion of diethylether in a slightly modified Rotations-Perforator after Ludwig for 1 h each at the natural pH, acidified with 0.1 N hydrochloric acid, and then made alkaline with 0.1 N sodium hydroxide solution. The combined organic phases were dried with anhydrous sodium sulphate, and the ether solution concentrated in a rotary evaporator. The remaining ether was blown off by ultrapure nitrogen, so that the volume of the raw extract was about 1 ml. This extract was liquid chromatographed on thoroughly precleaned silica gel (Kieselgel 60 Merck 0.04--0.063 mm) applying a low head pressure of nitrogen during elution. Fractionation was achieved in order of increasing polarities by gradient elution with n-hexane, cyclopentane, diethylether and methanol, monitored by UV-detection at 230 nm. Hydrocarbons were eluted with n-hexane in the first fraction. This hydrocarbon fraction was again chromatographed isokratically with n-hexane for separation of highly unsaturated hydrocarbons from aliphatics and mono- and diolefins. Aliquots
58
of the hydrocarbon fractions were injected into GC and/or GC--MS systems for identification of hydrocarbons. For GC and GC--MS conditions see Ehrhardt et al. (1980). Two internal standards, 1-tetradecene and 1-octadecene, which are known n o t to occur in seawater, were added to the samples at the start of the analytical procedure during homogenization, in order to evaluate the degree of recovery after the working-up procedure. Quantitation was achieved by peak area comparison with a standard n-alkane mixture by means of an electronic integrator. 1H-NMR-spectra were recorded on a Multikernresonanzspektrometer HX-90-R (Fa. Bruker Physic) at a frequency of 90 MHz, IR-spectrum on a Perkin-Elmer IR 421 spectrophotometer. Procedural blanks yielded some phthalates and other unidentified compounds in negligible amounts. RESULTS
All of the n-alkanes as well as pristane, phytane and squalene were identified by their Rf-values and mass-spectra by comparison with relevant standard substances. The c o m p o u n d s of the peak-groups which eluted mainly before the uneven numbered n-alkanes on a OV 101 stationary phase, showed the following behaviour: after hydrogenation only n-alkanes were left besides pristane and phytane, indicating that these hydrocarbons were neither branched nor alicychc. On a non-polar (OV 101) column they eluted before, but on a polar (Silar 7C) stationary phase after the uneven numbered n-alkane (see Figs. 4 and 5). The M+-peaks of their mass-spectra, although in the case of higher molecular weight c o m p o u n d s of low intensity, were two or four mass units below their neighbouring n-alkanes. Based on the analytical results these substances were identified as straight chain mono- and diolefins. In the second hquid--liquid chromatography the authors were able n o t only to separate peak No. 6 and squalene from the n-alkanes and n-alkenes b u t could also resolve peak No. 6 into three isomers (see Fig. 7). Separation was not good enough to result in pure fractions, b u t there are zones in the eluate of nearly pure compounds. Although the authors worked up combined samples in this analytical step, merely the amount of isomer III was sufficient to apply other analytical techniques for further structure elucidation in addition to coupled GC--MS. IR- and ~3C-NMR-spectra and the following data confirm a l l - c i s - h e n e i c o s a - 3 , 6, 9, 12, 15, 18-hexaene (HEH) as the structure of isomer III: Kovats Index on OV 101, 2042; on Silar 7C, 2270; A = 228. Hydrogenation converts the c o m p o u n d (and the remaining two isomers) into n-heneicosane. The 7 0 e V mass-spectrum corresponds with those reported in the literature (Lee et al., 1970), b u t in our spectra the M+-peak is missing. Perdeuteration, however, results in an M+-peak at m / z = 308. The difference of 12 mass units in the molecular weights of the products of hydrogenation and perdeuteration, respectively, means that the original c o m p o u n d contained six double bonds. The U V - s p e c ~ (n-hexane) showed no absorption above 210 nm, indicating the absence of conjugation.
59
IR-bands (film on NaCl-pellet) at 3 0 1 0 c m -1 (s) ( = C H - - ) ; 2 9 5 5 c m -1 (s), 2 8 7 0 c m -1 (m), 1 3 8 5 c m -1 ( m ) ( - - C H 3 ) ; 2 9 2 0 c m -1 (s), 2 8 4 5 c m -I (s), 1 4 4 0 - 1 3 7 0 c m -1 (m) (--CH2--); and a broad band centred on 7 1 0 c m -1 indicate an alkene with internal c/s-disubstituted, non-terminal double bonds. Absorption at 9 6 5 c m ~1 for trans-unsaturation was missing. The IH-NMRspectrum (CDC13, o given in ppm) shows resonance signals at o = 5.4(m, 1 2 - H , - - C H 2 - - C H = C H - - C H 2 - - ) , H o = 2.8 (m, 10-H, = C - - C H 2 - - C = ) , ~2L
H o = 2.05 (m, 4 - H , = C - - C H 2 - - C H 3) and o = 0.96 (t, 6-H, --CH2--CH3). When the protons at o = 0.96 were decoupled, the triplet disappeared and a singiet is seen instead; this demonstrates clearly that the methyl group is attached to a methylene group. Since there is no terminal double-bond, the c o m p o u n d in question is the symmetrical heneicosa-3, 6, 9, 12, 15, 18hexaene. The other t w o isomers have identical mass-spectra precisely matching the mass-spectrum of A-3 HEH. Heneicosane is formed upon hydrogena-
15
17
21
i$
\ Fig. 2. Gas chromatogram of the hydrocarbon fraction after the first liquid--liquid chromatographic separation, 5 m depth. Conditions: 30 m × 0.3 mm OV 101 glass capri. . . . • -I • -1 • • lazy, splitlessmjectlon(60s) a t 4 0 • C;40--250 o C a t 4 o C m m ;H 2 a t 2 . 5 m l m m ;rejector temperature 250 C; paper: 0.5 cmmin -1 . (Key: 6, n-heneicosahexaene; 15, n-pentadecane; 16, n-hexadecane; 17, n-heptadecane; 18, n-octadecane; IS, internal standard, either 1-tetradecene or 1-octadecene; Pr, pristane; Ph, phytane; 19, n-nonadecane; 20, n-eicosane; 21, n-heneicosane; 22, n-docosane; 23, n-tricosane; 24, n-tetracosane; 25, npentacosane; 26, n-hexacosane; 27, n-heptacosane; 28, n-octacosane; Sq, squalene; 29, n-nonacosane; 30, n-triacontane; 31, n-hentriacontane; m, mono-olefins; d, diolefins.)
16.5 17~5 18/19.5 20/21.5 22.5 25/26.5 16118.5 19120.5 20/21.5 21]22 5 23]26.5 28130.5 15117.5 17119.5 19/21:5 2!]22.5 25/29.5 29/31~5
6.3 4.5 7.3 5.8 6.0 5.9 6.4 6.1 7.5 4.1 5.4 4.2 3.2 n.d. 0.7 n.d. 0.6 2.6
n-C15
* Recovery rate o f n-octadeeene-1 and n-tetradecene-1 77% + 23% S.D.
AI.T. AI.T. AI.T. AI.T. AI.T. AI.T. A1.N. AI,N. Al.N. AI.N. A1.N. AI.N. O.P. O.P. O.P. O.P. O.P. O.P.
Sample (A1.T. = 5 m depth; O.P. = 1 m depth)
Concentrations of the four m o s t a b u n d a n t h y d r o c a r b o n s (ng1-1 seawater)*
TABLE I
6.5 4,7 6.2 4.4 7.1 4.7 5.2 5.9 8.7 4.3 7.0 4.4 3.8 2.1 3.1 0,4 2.4 3.0
n-C17 3.4 3.7 3.8 3.2 5.3 7.8 4.2 4.6 8.2 3.4 5.2 3.4 5.1 3.2 2.2 0.3 0.85 3.9
n-C21 46.2 52.4 63.5 58.6 72.4 87.5 22.6 67.7 97.3 57.4 72.4 43.8 79.6 65.4 84.6 56.3 14.7 76.2
n-C21:0(&-3 H E H )
O
61
tion, but we were not able to decide whether these two isomeric polyolefins are stereoisomers or double-bond position isomers. No significant differences in qualitative composition were observed in the general GC-pattern of aliphatic hydrocarbons between the samples taken at 1 and 5 m, respectively. The samples nearer the surface had lower concentrations of n-alkanes in the region of n-pentadecane and n-heptadecane and a slightly elevated content of the highly unsaturated compounds (Table I). The major compound in all samples was HEH. In Figs. 2 and 3, these differences are shown in typical gas chromatograms of samples from the two depths recorded after the first liquid chromatography. After repeated liquid chromatography, which separated the polyolefins, the remaining alkanes and rnono- and diolefins showed the following typical distribution (Figs. 4 and 5): a pronounced maximum with a clear predominance of uneven versus even carbon numbered n-alkanes in the first part of the chromatogram, start, ing with n-pentadecane, usually reaching a maximum at n-heptadecane (in some surface samples at n-heneicosane) and extending to nonacosane. In the S~
21
17
1
Fig. 3. G a s c h r o r a a t o g r a m o f t h e h y d r o c a r b o n f r a c t i o n a f t e r t h e f i r s t l i q u i d - - l i q u i d c h r o m a t o g r a p h y , 1 m d e p t h . C o n d i t i o n s a n d K e y as Fig. 2.
D--
Pr
Ph
Fig. 4. G a s c h r o m a t o g r a r n o f t h e f r a c t i o n o f n - a l k a n e s , m o n o - and diolef'ms after the second liquid--liquid chromatographic separation. Conditions: 6 0 m × 0.3 m m O V 101 glass c a p i l l a r y , sphtless " . . . rejection ( 6 0 s ) a t 4 0 o C; 4 0 - - 2 5 0 o C a t 4 o C r a m. _ 1; H2 a t 2 m l min-I ; i n j e c t o r temperature 2 5 0 o C; p a p e r : I c r n m m. _ 1. ( K e y as Fig. . 2.)
62 region with c a r b o n n u m b e r s higher than 22 the relative a m o u n t s o f n-alkanes d r o p m a r k e d l y and the e v e n / o d d p r e d o m i n a n c e is less p r o n o u n c e d . C o n c e n trations o f pristane and p h y t a n e are low, a n d the ratios p r i s t a n e / n - h e p t a d e cane and p h y t a n e / n - o c t a d e c a n e are less t h a n one. A g r o u p o f peaks a d j a c e n t to every o d d n u m b e r e d n-alkane starting with n - p e n t a d e c a n e and identified as m o n o - and diolefins is a characteristic feature which is m o s t o b v i o u s near n - h e p t a d e c a n e a n d n - n o n a c o s a n e (Fig. 6).
I?
'5
'
i
~
illi
19
20
2 2 23 24
t6
I 2 6 27,
2
ti
Fig. 5. Gas chromatogram of the fraction of n-alkanes, mono- and diolefins after the second liquid--liquid chromatographic separation, Conditions: 5 0 m × 0.3ram Silar 7c • . glass capillary; splitless injectlon (60s) at 60 o C; 60--260 o C at 4 o C m l ' n - I ; H2 at 1.5ml rain -1 ; injector temperature 250 oC; paper: 0.5 cm mm • -1 . (Key as Fig. • 2.) 17
tS
m m 19
le
m
i
Fig 6 Sectional blow up of the gas chromatogram o f Fig. 4. Conditions: 25 m × 0.3 m m SE '52" glass capillary; splitteu injection ( 6 0 s ) at 40~C; 40--260°C at 3 ° C m i n -1 ; H2 at 2 ml rain -1 , injector temperature 250 C; paper: 2 cm rain -11 (Key as Fig. 2.)
63 III
Sq II
Fig. 7. Gas chromatogram of the separated HEH-isomers and squalene. Conditions: . . . . . . O O
50m × 0.3mm Silar 7c glass capillary sphtless rejection (60s) at 60 C; 60--250 C at 4°Cmin-1; H2 at 2mlmin-i ; injector temperature 250°C; paper: 1 cmmin-1. (Key as
Fig. 2.)
The only branched chain hydrocarbons observed were pristane, phytane and squalene, the first two in relatively small amounts, the last comparable in amount to that of n-heptadecane. No aromatic hydrocarbons were detected. The four major constituents n-pentadecane, n-heptadecane, h-heneicosane and A-3 HEH were quantified. The mean recovery rate of 1-octadecene and 1-tetradecene was 77%. This was taken into account when calculating the data of Table I. DISCUSSION Since crude oil residues or petroleum-derived hydrocarbons are found in nearly all parts of the oceans, great efforts have been made to determine the portion of biogenic hydrocarbons. The hydrocarbon productivity of benthic and planktonic algae, especially, has been intensively surveyed, n-Pentadecane and n-heptadecane are reported to be the most abundant hydrocarbons of marine benthic algae (Clark and Blumer, 1967; Youngblood et al., 1971; Youngblood and Blumer, 1973), n-pentadecane prevailing in Phaeophyceae and Xanthophyceae, n-heptadecane in Rhodophyceae and Chlorophyceae. These classes of algae are also rich in highly unsaturated hydrocarbons, mainly with a straight chain of 19 or 21 carbon atoms and 5 or 6 double bonds. Unsaturated compounds are also predominant in marine planktonic algae (Blumer et al., 1971) with lesser contents of n-pentadecane and nheptadecane, as was found in a similar way by Lytle et al. (1979). Additionally they reported a group of phytadienes with R~-values between those of n-nonadecane and n-eicosane and a homologous series of n-alkenes, eluting 15--25 Kovats units after uneven carbon numbered n-alkanes starting with n-heptadecane and extending to n-pentacosane (stationary phase FFAP).
64 Besides this main source of aliphatic hydrocarbons, microorganisms may also contribute to the hydrocarbon pool in seawater (Oro et al., 1.967; Simoneit et al., 1979). A biogenic compound often referred to in the literature is HEH, whose structure and stereochemistry are often not fully elucidated. It was first detected in several varieties of marine algae and copepods by Blumer et al. (1970), isolated from the alga Fucus vesiculosus (Halsall and Hills, 1971), the diatom Skeletonema costaturn (Lee et al., 1970), and epipelic diatom populations in an estuary (Thompson and Eglinton, 1979). A more detailed assignment of the A-1 and A-3 HEH isomers to different classes of algae is given by Youngblood and Blumer (1973). They also found the corresponding pentaene, as well as C19-polyolefins with five and six double bonds. These authors and Lee and Loeblich (1971) also discussed the formation of A-3 HEH through decarboxylation of cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid. From the isoprenoid hydrocarbons only pristane was found in plant extracts (Clark and Blumer, 1967; Youngblood et al., 1971), whereas zooplankton and other marine animals contain pristane and phytane (Blumer et al., 1964; Avigan and Blumer, 1968) as well as the ubiquitous compound squalene. Phytane is said to occur in crude oil and sediments, but it was also detected in a pelagic Sargassum community (Burns and Teal, 1973) and unfiltered water samples in Bedford Basin (Gordon et al., 1978). Comparison between algae species cited in the literature as producing either HEH, n-Cls, n-C17 or olefins and those occurring in the Baltic shows that 42% of the benthic algae and 51% of the planktonic algae are also found in the Baltic; at least 71% of the benthic algae and 84% of the planktonic algae occur as the same variety (see Appendix). Although all these data originate from rather different hydrographical regimes, they have in common that the authors found more or less the complete spectrum of hitherto known aliphatic hydrocarbons released by benthic algae, phyto- and zooplankton and probably bacteria. In the water they appeared to be in a steady-state, renewed by biological activity and depleted by degradation and sedimentation. Particulate material should reflect this spectrum best, not only because phyto- and zooplankton themselves produce hydrocarbons and are part of the suspended material, but also because particles readily adsorb the dissolved but highly hydrophobic hydrocarbons. The authors' results support these assumptions quite well. The hydrocarbon spectrum of particulate material is thus characterized by: (I) a high content of n-alkanes belonging to a homologous series, which starts below n-dodecane, peaking with nheptadecane and with a pronounced uneven carbon number preference (CPI > 1); (2) a low content of higher molecular weight hydrocarbons with a CPI slightly greater than 1; (3) a predominance of HEH {three isomers); (4) a series of mono- and diolefins with uneven carbon numbers, most pronounced at C17, but also strong at nonacosane, and probably extended beyond Ca1 (the authors did not find, however, straight-chain polyolefins
65
with 19 carbon atoms); (5) a relativelylow concentration of the isoprenoid alkanes pristane and phytane, but a high concentration of squalene. The prevailing biological origin of the hydrocarbons in question is verified by the good agreement between the authors' findings, (1)--(5) above, and the data from the literature. A further support of their argument is the fact that their samples were taken from water containing a declining plankton bloom, which is evident from concentrations of nutrients, chlorophyll-a and D O C (A. Wenck, 1980, personal communication). The pattern of hydrocarbons remaining in the water from the plankton bloom is nearly as would be expected, according to literature data. This serves as a good proof for the relation between biological activityand hydrocarbon pattern. Most of the n-alkanes higher than tetracosane are probably derived from waxes of higher land plants (Eglinton and Hamilton, 1963; Kolattukudy and Walton, 1972). From the results of (2) above, it can be inferred that hydrocarbons coming from land run-off play a minor role as compared with the marine biogenic sources during a period of high biological activity,even in an area of high input of organic material by rivers. CONCLUSIONS
A correlation between particulate hydrocarbons and biological activity was already found by some authors. Goutx and Saliot (1980) found npentadecane and n-heptadecane predominating during plankton blooms in the Mediterranean; n-pentadecane partly made up 6 0 % of total hydrocarbons with concomitant high chlorophyll~ concentrations; at one station a significant correlation was observed between particulate n-alkanes and chlorophylls. A similar correlation between hydrocarbons and chlorophyll~ was stated by Zsolnay (1973) in an upwelling region off West Africa. The H E H seems to be a good indicator for phytoplankton activity.At the onset of a spring plankton bloom the concentrations of H E H in Bedford Basin increased sharply (Gordon et al., 1978), decreasing as the season progressed. The three diatoms Skeletonema eostatum, Detonula conferivacea and Thalassiosira nordenski~Idii, cultured in tank experiments, excreted mainly H E H during log phase growing, up to 0.6 -+0.13/~gmg -I dry weight of suspended material. After growth of the cultures had been stopped, concentrations dropped to zero within 12 weeks (Schulz and Quinn, 1977). Again, this demonstrates not only the relation between phytoplankton activityand concentration of H E H in suspended material, but also the replenishment of the highly unsaturated hydrocarbons in a natural population. The hydrocarbon pattern of dissolved or particulate matter is used as an indicator,whether or not this water is contaminated with petroleum hydrocarbons by oil or other anthropogenic sources, based upon the following premises: (1) A n uneven/even carbon number predominance, calculated for the range between n-dodecane and n-heneicosane, of CPI ~ 1 indicates anthro-
66
pogenic or geochemical origin; strong uneven carbon number predominance, CPI ¢ 1, indicates a biogenic origin of the hydrocarbon mixture. (2) A high concentration of n-alkanes in the range between ~-heneicosane and n-triacontane relative to the concentration in the range between n-dodecane and n-heneicosane as well as a high content of an unresolved complex mixture (UCM), strongly indicates crude oil or petroleum-derived hydrocarbons. (3) The absence or presence of aromatic and alicyclic hydrocarbons; the latter indicating pollution. (4) The phytane/pristane ratio as well as the ratios of pristane/n-heptadecane and phytane/n-octadecane; high ratios make pollution influence very likely. Comparing the authors' results with this set of conditions, the area of investigation may be classified as non-polluted in the above-mentioned sense. In addition they are evidence for biological activity unimpeded by pollution. Therefore, in order to estimate the degree of pollution by non-biogenic hydrocarbons and the effect of this pollution on life processes, the following two items should also be considered: (5) The content of HEH (and probably related highly unsaturated Cl9 and C21 hydrocarbons) and their relation to the sum of the other hydrocarbons. (6) The absence or presence of mono- and diolefins with carbon numbers, starting with C17 and extending b e y o n d C,9. To prove the applicability of this hypothesis, investigations on the seasonal dependence of these parameters should be continued. ACKNOWLEDGEMENTS
The authors thank Christian Wolff for recording and assistance in interpretation of the NMR-spectra and Manfred Ehrhardt for valuable discussions. This research was supported by grant No. MFU 0 5 0 6 / T V 1 of the Bundesministerium f~/r Forschung and Technologie.
67 APPENDIX List of algae species cited in literature to produce either HEH, n-Cls, n-C17, olefins and their occurrence in the Baltic Sea according to Pankow (1971 ) Class
Order
Variety/species
Occurrence
References*
Species
Variety only
Ulva lactuca Monostroma sp.
+ --
+ +
I, 5 1, 8
E n t e r o m o r p h a linza Enteromorpha compressa S p o n g a m o r p h a arcta H a l i m e d a sp. Halimeda discoidea Caulerpa sertularoides C o d i u m fragile Codium repens Codium cardioniarum Dunaliella tertiolata Prasiola stipitata Cystod~ctyon pavonium P l a t y m o n a s sp.
÷ @ --
+ q+
1 5 1, 8, 5
Benthic algae Clorophycea
Ulotrichales
Cladophorales Siphonales
Rhodophyceae
Volvocales Schizogoniales Siphonocladales Pyramimonadales Bangialea Cryptonemiales
Gigartinales
Rhodymeniales Ceramiales
Phaeophyceae
Fucales
Laminariales
Porphyra leucosticta D u m o n t i a inerassata Kallymenia perforata H a l y m e n i a sp. P e y s s o n n e l i a rubia Corallina o f f i c i n a l i s C h o n d r u s crispus Gracilaria e y l i n d r i c a Gracilaria b l o d g e t t i i Gracilaria m a m m i l a r i s Euchenma isoforme Euchenma compressa Rhodymenia palmata Rhody menia conferoides Ceramium rubrum P o l y s i p h o n i a ureeolata L a u r e n t i a corraUopsi~ L a u r e n t i a intricatu Ascophyllum nodosum F u c u s sp. Fucus distichus F u e u s spiralis F u c u s vesiculosus Sargassum sp. Agarum cribrosum Chorda filum Chorda tomentosa L a m i n a r i a digitata L a m i n a r i a saccarina L a m i n a r i a agardlin
--
--
6
---
---
6 6
--
--
10, 6, 5
---+ --
---+ --
6 6 3, 8 1 6
-+
+ +
10 I, 3, 5
+ --
+ --
5 6
--
+
6
-+ + -----+ -+ + -+
-+ + + + + --+ + + + --4-
6 4 5, 4 6 6 6 6 6
---
+ +
-+
+ +
--
+
-+ + + + --
-+ + + -b +
5, 4 1 5
5, 4 6 6
1, 8, 5, 4 4 1, 8, 5 5
1, 2, 8, 5 4 4 5 5
1, 5, 4 1, 8 5
68 APPENDIX (continued) Class
Order
Desmarestiales Ectoearpales
Punetariales Dictyotales Chordariales
Variety/species
Desmarestia viridis Ectocarpus fnsciculatus Ectocarpus siliculosus PilayeUa littorales Streblonerna oligosporum Scytosiphon lomentaria Punctaria latifolia Dictyota dichotoma Chordaria flagelliform is Leathesia difformis
()ccurrence
l~e ferenees*
Species
Variety only
~
~ ~
I , :~ I, i
4 ~
~ + ÷ ~-
10 I, 5 lO 1,8, 5
+
~
-+ ~
+ ~
5 6 5 5
÷ q-
+ +
3, 8
-
+ +
"
+
10 3, 8, 10 10 3, 8, 4
Planktonic algae Dinophyceae
Cryptophyceae
Euglenaphyceae Cyanophyceae Ptuinophyceae Desmophycea Cachoniaceae BaciUariophyceae (Diatomaphyceae)
Peridinales
Cryptomonadales
Euglenales Nostocales
Prorocentrales Rhizosoleniales
Coscinodiscales
Biddulphiales Naviculales Chrysophyceae
Xanthophyceae
Chrysomonadales
Chrysocapmdes Heterotrichales
Gongaulax polyedra Peridinium trochoideum Peridinum sociale G y m n o d i n i u m splendens Cryptomonns ovata Cryptomonas sp. R h o d o m o n a s lens Eutreptia viridis Euglena gracilis Synechoccus baciUaris Oscillatoria woronichinii Pyraminonns sp. Exuviella cassubica Cachonina niei Detonula conferivace Dity lum brightwellii Lauderia borealis Rhizosolenia setigera Thalassiosira fluviatilis Thalassiosira nordenski61dii S k e l e t o n e m a costatum Cyclotella nana Chaetoceros curvisetus Cy lindro theca fusiformis Navicula polliculosa Prymnesium parvum Syracosphera carterae Isochrysi~ galbana Coccolithus huxlegi Phaeocystis poncheti Tribonema aequale
d-
3, 8, 10
~
10
+
~
3, 8, 10
-
+ +
10 3 3
--
--
3
+
+
10
+
+
9
q+
+ +
3, 8, 10 3, 8, 10
+ + +
+ + +
3 3, 8 9
+
+
3, 8, 9, 1O, 7, 4
........
10
+
3
l-
+
+ Jr
+ + + -
3, 10 10
-t +
10
10 10, 4
+
3, 8, 10
~+ --
3, 8 3, 8 3, 8
* 1, Younllblood and Blumer (1973); 2, Halsall and Hill~ (1971); 3, Blumer et al. (1970); 4, Clark and Blumer (1967); 5, Youngblood et al. (1971); 6, Lytle et al. (1979); 7, Lee et al. (1970); 8, Blumer et al. (1971); 9, Schultz and Quinn (1977); 10, Lee and Loeblich (1971); 11, Pankow (1971).
69 REFERENCES Avigan, J. and Blumer, M., 1968. O n the origin of pristane in marine organisms. J. Lipochim. Res., 9: 350--352. Blumer, M., Mullin, M.M. and Thomas, D.W., 1964. Pristane in the marine environment. Helgol~inder wiss. Meeresunters., 10: 187--201. Blumer, M., Mullin, M.M. and Guillard, R.R.L., 1970. A polyunsaturated hydrocarbon (3, 6, 9, 12, 15, 18-heneicosahexaene) in the marine food web. Mar. Biol., 6: 226--235. Blumer, M., Guillard, R.R.L. and Chase, T., 1971. Hydrocarbons of marine phytoplankton. Mar. Biol., 8: 183--189. Burns, K.A. and Teal, M., 1973. Hydrocarbons in the pelagic Sargassum community. Deep-Sea Res., 20: 207--211. Cauwet, G., 1978. Organic chemistry of sea water particulates. Concepts and developmerits. Oceanol. Acta, 1: 99--105. Clark, R.C. and Blumer, M., 1967. Distribution of n-paraffins in marine organisms and sediment. Limnol. Oceanogr., 12: 79--87. Crisp, P.T., Brenner, S., Venkatesan, M.I., Ruth, E. and Kaplan, I.R., 1979. Organic chemical characterization of sediment-trap particulates from San Nicolas, Santa Barbara, Santa Monica, and San Pedro Basins, California. Geochim. Cosmochim. Acta, 43: 1791--1801. Derenbach, J., 1969. Partikul~re Substanz und Plankton an Hand chemischer und biologischer Daten, gemessen in den oberen Wasserschichten des Gotland-Tief im Mai 1968. Kiel. Meeresforsch., 25: 279--289. Eglinton, G. and Hamilton, R.J., 1963. The distribution of alkanes. In: T. Swain (Editor), Chemical Plant Taxonomy. Academic Press, New York, NY, pp. 187--217. Ehrhardt, M., 1969. The particulate organic carbon and nitrogen, and the dissolved organic carbon in the Gotland Deep in May 1968. Kiel. Meeresforsch., 25: 71--80. Ehrhardt, M., 1978. An automatic sampling buoy for the accumulation of dissolved and particulate organic material from seawater. Deep-Sea Res., 25: 119--126. Ehrhardt, M., Osterroht, C. and Petrick, G., 1980. Fatty-acid methyl esters dissolved in seawater and associated with suspended particulate material. Mar. Chem., 10: 67--76. Gearing, P.J., Gearing, J.N., Pruell, R.J., Wade, T.L. and Quinn, J.G., 1980. Partititioning of No. 2 Fuel Oil in controlled estuarine ecosystems. Sediments and suspended particulate matter. Environ. Sci. Technol., 14: 1129--1136. Gordon, D.C., Keizer, J.P.D. and Dale, J., 1978. Temporal variations and probable origins of hydrocarbons in the water column of Bedford Basin, Nova Scotia. Estuarine Coastal Mar. Sci., 7,243--256. Goutx, M. and Saliot, A., 1980. Relationship between dissolved and particulate fatty acids and hydrocarbons, chlorophyll-a and zooplankton biomass in Villefranche Bay/ Mediterranean. Mar. Chem., 8: 299--318. Halsall, T.G. and Hills, I.R., 1971. Isolation of Heneicosa-1, 6, 9, 12, 15, 18-hexaene and -1,6, 9,12, 15-pentaene from the alga Fucus vesiculosus. Chem. Commun., 448--449. Kolattukudy, P.E. and Walton, T.J., 1972. The biochemistry of plant cuticular lipids. In: R.T. Holman (Editor), Progress in the Chemistry of Fats and Other Lipids. Pergamon Press, Oxford, Vol. 13, pp. 121--175. Kremling, K., Osterroht, C. and Wenck, A., 1981. Investigations on dissolved copperorganic substances in Baltic waters. Mar. Chem., 10: 209--219. Lee, R.F. and Loeblich III, A.R., 1971. Distribution of 21:6 hydrocarbon and its relationship to 22:6 fatty acid in algae. Phytochemistry, 10: 593--602. Lee, R.F., Nevenzel, J.C., Paffenh~fer, G.A., Benson, A.A., Patton, S. and Kavannagh, T.E., 1970. A unique hexaene from a diatom (Skeletonema costatum). Biochim. Biophys. Acta, 202: 386--388. Lytle, T.F. and Lytle, J.S., 1979. Sediment hydrocarbons near an oil rig. Estuarine Coastal Mar. Sci., 9: 319--330.
70 Lytle, J.S., Lytle, T.F., Gearing, J.N. and Gearing, P.J., 1979. Hydrocarbons in benthic algae from the eastern Gulf of Mexico. Mar. Biol., 51 : 279--288. Morris, B.F., Butler, J.N., Sleeter T.D. and Cadwallader, J., 1977. Particulate hydrocarbon material in ocean waters. Rapp. P.V. Rdun. Cons. Int. Explor. Mer., 171: 107--116. Oro, J., Tornabene, T.G., Nooner, D.W. and Gelpi, E , 1967. Aliphatic hydrocarbons and fatty acids of some marine and freshwater microorganisms. J. Bacteriol. 93: 1811--1818. Pankow, H., 1971. Algenflora der Ostsee, Bd. I, II, III. Gustav Fischer Vertag, Stuttgart. Prahl, F.G. and Carpenter, R., 1979. The role of zooplankton fecal pellets in the sedimentation of polycyclic aromatic hydrocarbons in Dabob Bay, Washington. Geochim. Cosmochim. Acta, 43: 1959--1972. Schultz, D.M. and Quinn, J.G., 1977. Suspended material in Narragansett Bay: fatty acid and hydrocarbon composition. Org. Geochem., 1: 27--36. Simoneit, B.R.T., Mazurek, M.A. and Brenner, S., 1979. Organic geochemistry of recent sediments from Guaymas Basin, Gulf of California. Deep-Sea Res., 26A: 879--891. Thompson, S. and Eglinton, G., 1979. The presence of pollutant hydrocarbons in esturine epipelic diatom populations. II. Diatom Slimes. Estuarine Coastal Mar. Sci., 8: 75 --86. Wangersky, P.J., 1974. Particulate organic carbon: sampling variability. Limnol. Oceanogr., 19: 980--984. Wangersky, P.J., 1976. Particulate organic carbon in the Atlantic and Pacific Oceans. Deep-Sea Res., 23: 457--465. Wade, T.L. and Quinn, J.G., 1980. Incorporation, distribution and fate of saturated petroleum hydrocarbons in sediments from a controlled marine ecosystem. Mar. Environ. Res., 3: 15--33. Youngblood, W.W. and Blumer, M., 1973. Alkanes and alkenes in marine benthic algae. Mar. Biol., 21: 163--172. Youngblood, W.W., Blumer, M., Guillard, R.L. and Fiore, F., 1971. Saturated and unsaturated hydrocarbons in marine benthic algae. Mar. Biol., 8: 190--201. Zsolnay, A., 1971. A preliminary study of the dissolved hydrocarbons and hydrocarbons on particulate material in the Gotland Deep of the Baltic. Kiel. Meeresforsch., 27: 129--134. Zsolnay, A., 1973. Hydrocarbon and chlorophyll: correlation in the upwelling region off West Africa. Deep-Sea Res., 20: 923--925.
55
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
A LI P HATI C HY D R O C A R B O N S IN P A R T I C U L A T E M A T T E R FROM T H E BALTIC SEA CHR. OSTERROHT and G. PETRICK Institut fiir Meereskunde an der Uniuersit;it Kiel, Diisternbrooker Weg 20, D-2300 Kiel (G.F.R.)
(Received March 20, 1981; revision accepted September 24, 1981) ABSTRACT Osterroht, Chr. and Petrick, G., 1982. Aliphatic hydrocarbons in particulate matter from the Baltic Sea. Mar. Chem., 11: 55--70. During May 1980, particulate matter was collected at an anchor station from different depths by two methods. In the separated hydrocarbon fraction the most abundant component was the highly unsaturated hydrocarbon n-heneicosahexaene (HEH). Also present was a series of mono- and di-unsaturated olefinic hydrocarbons with an uneven number of carbon atoms, starting with n-ClT. The alkane pattern was characterized by a maximum in the range between n-pentadeeane and n-heptadecane, with a strong uneven to even carbon number predominance. The peculiar composition of the aliphatic hydrocarbons was compared with literature data given for aliphatic hydrocarbons, whether produced by different marine organisms or derived from crude oil of other anthropogenic sources. A suggestion is made for using a set of conditions as indicators for pollution by fossil hydrocarbons. INTRODUCTION It is generally agreed t h a t particles having a diameter of m ore than 0.45 #m m ay be considered as particulate m a t t e r and all material with a smaller diameter is said to be dissolved, either colloidally or in molecular dispersion. Particulate m a t t e r m a y be divided into t w o categories: living organisms and detrital material (Cauwet, 1978). Most planktonic organisms are mainly f o u n d in the euphoric layer which in the Baltic Sea extends to 20 m dept h on average and is subject to a rapid and t h o r o u g h turnover. Detritus, however, is very stable with respect to minerals, remainders of living organisms and some organic com pounds , be i t adsorbed or incorporated. Therefore, the c o n c e n t r a t i o n of particulate organic carbon in the euphoric zone is n o t only higher by a factor of 5--10 relative to concentrations at greater dept h (Wangersky, 1974, 1976), but t he pat t er n of organic molecules in surface water particulate m a t t e r also reflects the state o f the water b o d y in question, i.e. biological activity as well as atmospheric input, land run off, etc. The relatively stable h y d r o c a r b o n s associated with suspended material, especially, are capable of reflecting the state of the environment, as shown by m a n y authors. F r o m gas chromatographic h y d r o c a r b o n patterns o f particles collected in sediment.traps (Crisp et al., 1979) and from filtered particulate material (Morris et al., 1977), biosynthetic origin as well as pollution, seepage or
0304-4203/82/0000-0000/$02.75 01982 Elsevier Scientific Publishing Company
56 other chemogenic origins can be inferred. In addition, several authors have demonstrated that particulate matter is a vehicle for the transport of hydrocarbons and renders the underlying sediments an indicator of processes in the water column (Lytle and Lytle, 1979). In tank experiments it was shown that aliphatics of a crude oil were predominantly associated with particulate matter and deposited on sediments, leading to a gas chromatographic pattern from the sediment, typical of the aliphatics of the crude oil. It was also confirmed that the extent of adsorption is inversely proportional to the solubility of the compounds in water, which means that the more soluble aromatics were depleted on suspended matter. Partition concentrations of the crude oil to water, particulate matter and sediments were the same as those found in the Baltic Sea after the Tsesis oil spill (Gearing et al., 1980; Wade and Quinn, 1980). On the other hand, polycyclic aromatic hydrocarbons (PAH), if sufficiently concentrated in the water, are also transported by suspended material to the sediments, as shown by Prahl and Carpenter (1979) by the remarkable similarity of PAH patterns in plankton, trap material and sediments. The Baltic Sea is a land-locked sea characterized by high river inputs of terrestrial material and a typical b o t t o m topography, characterized by three basins, separated by sills. The Gotland Deep (the area of this investigation) is the largest and deepest of these basins. Nevertheless, the distribution of particulate matter in the Gotland Deep follows the previously mentioned pattern of Wangersky: particulate organic carbon is most highly concentrated in the surface water, followed by a maximum of atomic ratio N/Cpart" at a b o u t 10 m, indicating that primary production is most intensive in this zone (Ehrhardt, 1969). Data of simultaneously investigated plankton patterns support this conclusion (Derenbach, 1969). Total concentrations of hydrocarbons, dissolved in water and adsorbed onto particulate material in the Gotland Deep, were measured by Zsolnay (1971) with a CHN-analyzer as hydrocarbon-derived organic carbon. The aim of this work was to evaluate the content of hydrocarbons on particulate matter and to compare the reresults with other data. Materials and methods
During May 1980, at an anchor station in the Gotland Sea (see Fig. 1 for geographic localities), samples were taken either by an automatic sampling b u o y (6 samples) from 1 m depth or from a depth of approximately 5 m by a ship-borne pumping system permitting pH control of the sampled water (12 samples). For technical details of sampling devices see Ehrhardt (1978) and Kremling et al. (1981). Material collected on filters by both sampling procedures was used for this investigation. All solvents were analytical grade, redistilled in an all-glass still over a 1 5 0 c m column filled with glass spirals; water was purified with a Milli-Q system by Millipore, including mixed-bed exchanger, activated charcoal and
57
Fig. 1. Map of the area of investigation. 0.2 tzm filter. Filters were Schleicher & Schull glass-fibre filters No. 6, heated in a drying cabinet at 300°C in an oxygen atmosphere. After filtration (for one sample about 4001 of seawater in the buoy system and about 2001 of seawater in the pH-controlled procedure) the filters were homogenized with diethylether by means of a Cenco high-speed homogenizer, resulting in a first extraction of lipophilic organic material under mild thermal conditions. After phase separation, the ether was decanted and the remaining homogenate was suspended in water free of organic carbon. The aqueous phase was extracted three times with an additional portion of diethylether in a slightly modified Rotations-Perforator after Ludwig for 1 h each at the natural pH, acidified with 0.1 N hydrochloric acid, and then made alkaline with 0.1 N sodium hydroxide solution. The combined organic phases were dried with anhydrous sodium sulphate, and the ether solution concentrated in a rotary evaporator. The remaining ether was blown off by ultrapure nitrogen, so that the volume of the raw extract was about 1 ml. This extract was liquid chromatographed on thoroughly precleaned silica gel (Kieselgel 60 Merck 0.04--0.063 mm) applying a low head pressure of nitrogen during elution. Fractionation was achieved in order of increasing polarities by gradient elution with n-hexane, cyclopentane, diethylether and methanol, monitored by UV-detection at 230 nm. Hydrocarbons were eluted with n-hexane in the first fraction. This hydrocarbon fraction was again chromatographed isokratically with n-hexane for separation of highly unsaturated hydrocarbons from aliphatics and mono- and diolefins. Aliquots
58
of the hydrocarbon fractions were injected into GC and/or GC--MS systems for identification of hydrocarbons. For GC and GC--MS conditions see Ehrhardt et al. (1980). Two internal standards, 1-tetradecene and 1-octadecene, which are known n o t to occur in seawater, were added to the samples at the start of the analytical procedure during homogenization, in order to evaluate the degree of recovery after the working-up procedure. Quantitation was achieved by peak area comparison with a standard n-alkane mixture by means of an electronic integrator. 1H-NMR-spectra were recorded on a Multikernresonanzspektrometer HX-90-R (Fa. Bruker Physic) at a frequency of 90 MHz, IR-spectrum on a Perkin-Elmer IR 421 spectrophotometer. Procedural blanks yielded some phthalates and other unidentified compounds in negligible amounts. RESULTS
All of the n-alkanes as well as pristane, phytane and squalene were identified by their Rf-values and mass-spectra by comparison with relevant standard substances. The c o m p o u n d s of the peak-groups which eluted mainly before the uneven numbered n-alkanes on a OV 101 stationary phase, showed the following behaviour: after hydrogenation only n-alkanes were left besides pristane and phytane, indicating that these hydrocarbons were neither branched nor alicychc. On a non-polar (OV 101) column they eluted before, but on a polar (Silar 7C) stationary phase after the uneven numbered n-alkane (see Figs. 4 and 5). The M+-peaks of their mass-spectra, although in the case of higher molecular weight c o m p o u n d s of low intensity, were two or four mass units below their neighbouring n-alkanes. Based on the analytical results these substances were identified as straight chain mono- and diolefins. In the second hquid--liquid chromatography the authors were able n o t only to separate peak No. 6 and squalene from the n-alkanes and n-alkenes b u t could also resolve peak No. 6 into three isomers (see Fig. 7). Separation was not good enough to result in pure fractions, b u t there are zones in the eluate of nearly pure compounds. Although the authors worked up combined samples in this analytical step, merely the amount of isomer III was sufficient to apply other analytical techniques for further structure elucidation in addition to coupled GC--MS. IR- and ~3C-NMR-spectra and the following data confirm a l l - c i s - h e n e i c o s a - 3 , 6, 9, 12, 15, 18-hexaene (HEH) as the structure of isomer III: Kovats Index on OV 101, 2042; on Silar 7C, 2270; A = 228. Hydrogenation converts the c o m p o u n d (and the remaining two isomers) into n-heneicosane. The 7 0 e V mass-spectrum corresponds with those reported in the literature (Lee et al., 1970), b u t in our spectra the M+-peak is missing. Perdeuteration, however, results in an M+-peak at m / z = 308. The difference of 12 mass units in the molecular weights of the products of hydrogenation and perdeuteration, respectively, means that the original c o m p o u n d contained six double bonds. The U V - s p e c ~ (n-hexane) showed no absorption above 210 nm, indicating the absence of conjugation.
59
IR-bands (film on NaCl-pellet) at 3 0 1 0 c m -1 (s) ( = C H - - ) ; 2 9 5 5 c m -1 (s), 2 8 7 0 c m -1 (m), 1 3 8 5 c m -1 ( m ) ( - - C H 3 ) ; 2 9 2 0 c m -1 (s), 2 8 4 5 c m -I (s), 1 4 4 0 - 1 3 7 0 c m -1 (m) (--CH2--); and a broad band centred on 7 1 0 c m -1 indicate an alkene with internal c/s-disubstituted, non-terminal double bonds. Absorption at 9 6 5 c m ~1 for trans-unsaturation was missing. The IH-NMRspectrum (CDC13, o given in ppm) shows resonance signals at o = 5.4(m, 1 2 - H , - - C H 2 - - C H = C H - - C H 2 - - ) , H o = 2.8 (m, 10-H, = C - - C H 2 - - C = ) , ~2L
H o = 2.05 (m, 4 - H , = C - - C H 2 - - C H 3) and o = 0.96 (t, 6-H, --CH2--CH3). When the protons at o = 0.96 were decoupled, the triplet disappeared and a singiet is seen instead; this demonstrates clearly that the methyl group is attached to a methylene group. Since there is no terminal double-bond, the c o m p o u n d in question is the symmetrical heneicosa-3, 6, 9, 12, 15, 18hexaene. The other t w o isomers have identical mass-spectra precisely matching the mass-spectrum of A-3 HEH. Heneicosane is formed upon hydrogena-
15
17
21
i$
\ Fig. 2. Gas chromatogram of the hydrocarbon fraction after the first liquid--liquid chromatographic separation, 5 m depth. Conditions: 30 m × 0.3 mm OV 101 glass capri. . . . • -I • -1 • • lazy, splitlessmjectlon(60s) a t 4 0 • C;40--250 o C a t 4 o C m m ;H 2 a t 2 . 5 m l m m ;rejector temperature 250 C; paper: 0.5 cmmin -1 . (Key: 6, n-heneicosahexaene; 15, n-pentadecane; 16, n-hexadecane; 17, n-heptadecane; 18, n-octadecane; IS, internal standard, either 1-tetradecene or 1-octadecene; Pr, pristane; Ph, phytane; 19, n-nonadecane; 20, n-eicosane; 21, n-heneicosane; 22, n-docosane; 23, n-tricosane; 24, n-tetracosane; 25, npentacosane; 26, n-hexacosane; 27, n-heptacosane; 28, n-octacosane; Sq, squalene; 29, n-nonacosane; 30, n-triacontane; 31, n-hentriacontane; m, mono-olefins; d, diolefins.)
16.5 17~5 18/19.5 20/21.5 22.5 25/26.5 16118.5 19120.5 20/21.5 21]22 5 23]26.5 28130.5 15117.5 17119.5 19/21:5 2!]22.5 25/29.5 29/31~5
6.3 4.5 7.3 5.8 6.0 5.9 6.4 6.1 7.5 4.1 5.4 4.2 3.2 n.d. 0.7 n.d. 0.6 2.6
n-C15
* Recovery rate o f n-octadeeene-1 and n-tetradecene-1 77% + 23% S.D.
AI.T. AI.T. AI.T. AI.T. AI.T. AI.T. A1.N. AI,N. Al.N. AI.N. A1.N. AI.N. O.P. O.P. O.P. O.P. O.P. O.P.
Sample (A1.T. = 5 m depth; O.P. = 1 m depth)
Concentrations of the four m o s t a b u n d a n t h y d r o c a r b o n s (ng1-1 seawater)*
TABLE I
6.5 4,7 6.2 4.4 7.1 4.7 5.2 5.9 8.7 4.3 7.0 4.4 3.8 2.1 3.1 0,4 2.4 3.0
n-C17 3.4 3.7 3.8 3.2 5.3 7.8 4.2 4.6 8.2 3.4 5.2 3.4 5.1 3.2 2.2 0.3 0.85 3.9
n-C21 46.2 52.4 63.5 58.6 72.4 87.5 22.6 67.7 97.3 57.4 72.4 43.8 79.6 65.4 84.6 56.3 14.7 76.2
n-C21:0(&-3 H E H )
O
61
tion, but we were not able to decide whether these two isomeric polyolefins are stereoisomers or double-bond position isomers. No significant differences in qualitative composition were observed in the general GC-pattern of aliphatic hydrocarbons between the samples taken at 1 and 5 m, respectively. The samples nearer the surface had lower concentrations of n-alkanes in the region of n-pentadecane and n-heptadecane and a slightly elevated content of the highly unsaturated compounds (Table I). The major compound in all samples was HEH. In Figs. 2 and 3, these differences are shown in typical gas chromatograms of samples from the two depths recorded after the first liquid chromatography. After repeated liquid chromatography, which separated the polyolefins, the remaining alkanes and rnono- and diolefins showed the following typical distribution (Figs. 4 and 5): a pronounced maximum with a clear predominance of uneven versus even carbon numbered n-alkanes in the first part of the chromatogram, start, ing with n-pentadecane, usually reaching a maximum at n-heptadecane (in some surface samples at n-heneicosane) and extending to nonacosane. In the S~
21
17
1
Fig. 3. G a s c h r o r a a t o g r a m o f t h e h y d r o c a r b o n f r a c t i o n a f t e r t h e f i r s t l i q u i d - - l i q u i d c h r o m a t o g r a p h y , 1 m d e p t h . C o n d i t i o n s a n d K e y as Fig. 2.
D--
Pr
Ph
Fig. 4. G a s c h r o m a t o g r a r n o f t h e f r a c t i o n o f n - a l k a n e s , m o n o - and diolef'ms after the second liquid--liquid chromatographic separation. Conditions: 6 0 m × 0.3 m m O V 101 glass c a p i l l a r y , sphtless " . . . rejection ( 6 0 s ) a t 4 0 o C; 4 0 - - 2 5 0 o C a t 4 o C r a m. _ 1; H2 a t 2 m l min-I ; i n j e c t o r temperature 2 5 0 o C; p a p e r : I c r n m m. _ 1. ( K e y as Fig. . 2.)
62 region with c a r b o n n u m b e r s higher than 22 the relative a m o u n t s o f n-alkanes d r o p m a r k e d l y and the e v e n / o d d p r e d o m i n a n c e is less p r o n o u n c e d . C o n c e n trations o f pristane and p h y t a n e are low, a n d the ratios p r i s t a n e / n - h e p t a d e cane and p h y t a n e / n - o c t a d e c a n e are less t h a n one. A g r o u p o f peaks a d j a c e n t to every o d d n u m b e r e d n-alkane starting with n - p e n t a d e c a n e and identified as m o n o - and diolefins is a characteristic feature which is m o s t o b v i o u s near n - h e p t a d e c a n e a n d n - n o n a c o s a n e (Fig. 6).
I?
'5
'
i
~
illi
19
20
2 2 23 24
t6
I 2 6 27,
2
ti
Fig. 5. Gas chromatogram of the fraction of n-alkanes, mono- and diolefins after the second liquid--liquid chromatographic separation, Conditions: 5 0 m × 0.3ram Silar 7c • . glass capillary; splitless injectlon (60s) at 60 o C; 60--260 o C at 4 o C m l ' n - I ; H2 at 1.5ml rain -1 ; injector temperature 250 oC; paper: 0.5 cm mm • -1 . (Key as Fig. • 2.) 17
tS
m m 19
le
m
i
Fig 6 Sectional blow up of the gas chromatogram o f Fig. 4. Conditions: 25 m × 0.3 m m SE '52" glass capillary; splitteu injection ( 6 0 s ) at 40~C; 40--260°C at 3 ° C m i n -1 ; H2 at 2 ml rain -1 , injector temperature 250 C; paper: 2 cm rain -11 (Key as Fig. 2.)
63 III
Sq II
Fig. 7. Gas chromatogram of the separated HEH-isomers and squalene. Conditions: . . . . . . O O
50m × 0.3mm Silar 7c glass capillary sphtless rejection (60s) at 60 C; 60--250 C at 4°Cmin-1; H2 at 2mlmin-i ; injector temperature 250°C; paper: 1 cmmin-1. (Key as
Fig. 2.)
The only branched chain hydrocarbons observed were pristane, phytane and squalene, the first two in relatively small amounts, the last comparable in amount to that of n-heptadecane. No aromatic hydrocarbons were detected. The four major constituents n-pentadecane, n-heptadecane, h-heneicosane and A-3 HEH were quantified. The mean recovery rate of 1-octadecene and 1-tetradecene was 77%. This was taken into account when calculating the data of Table I. DISCUSSION Since crude oil residues or petroleum-derived hydrocarbons are found in nearly all parts of the oceans, great efforts have been made to determine the portion of biogenic hydrocarbons. The hydrocarbon productivity of benthic and planktonic algae, especially, has been intensively surveyed, n-Pentadecane and n-heptadecane are reported to be the most abundant hydrocarbons of marine benthic algae (Clark and Blumer, 1967; Youngblood et al., 1971; Youngblood and Blumer, 1973), n-pentadecane prevailing in Phaeophyceae and Xanthophyceae, n-heptadecane in Rhodophyceae and Chlorophyceae. These classes of algae are also rich in highly unsaturated hydrocarbons, mainly with a straight chain of 19 or 21 carbon atoms and 5 or 6 double bonds. Unsaturated compounds are also predominant in marine planktonic algae (Blumer et al., 1971) with lesser contents of n-pentadecane and nheptadecane, as was found in a similar way by Lytle et al. (1979). Additionally they reported a group of phytadienes with R~-values between those of n-nonadecane and n-eicosane and a homologous series of n-alkenes, eluting 15--25 Kovats units after uneven carbon numbered n-alkanes starting with n-heptadecane and extending to n-pentacosane (stationary phase FFAP).
64 Besides this main source of aliphatic hydrocarbons, microorganisms may also contribute to the hydrocarbon pool in seawater (Oro et al., 1.967; Simoneit et al., 1979). A biogenic compound often referred to in the literature is HEH, whose structure and stereochemistry are often not fully elucidated. It was first detected in several varieties of marine algae and copepods by Blumer et al. (1970), isolated from the alga Fucus vesiculosus (Halsall and Hills, 1971), the diatom Skeletonema costaturn (Lee et al., 1970), and epipelic diatom populations in an estuary (Thompson and Eglinton, 1979). A more detailed assignment of the A-1 and A-3 HEH isomers to different classes of algae is given by Youngblood and Blumer (1973). They also found the corresponding pentaene, as well as C19-polyolefins with five and six double bonds. These authors and Lee and Loeblich (1971) also discussed the formation of A-3 HEH through decarboxylation of cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid. From the isoprenoid hydrocarbons only pristane was found in plant extracts (Clark and Blumer, 1967; Youngblood et al., 1971), whereas zooplankton and other marine animals contain pristane and phytane (Blumer et al., 1964; Avigan and Blumer, 1968) as well as the ubiquitous compound squalene. Phytane is said to occur in crude oil and sediments, but it was also detected in a pelagic Sargassum community (Burns and Teal, 1973) and unfiltered water samples in Bedford Basin (Gordon et al., 1978). Comparison between algae species cited in the literature as producing either HEH, n-Cls, n-C17 or olefins and those occurring in the Baltic shows that 42% of the benthic algae and 51% of the planktonic algae are also found in the Baltic; at least 71% of the benthic algae and 84% of the planktonic algae occur as the same variety (see Appendix). Although all these data originate from rather different hydrographical regimes, they have in common that the authors found more or less the complete spectrum of hitherto known aliphatic hydrocarbons released by benthic algae, phyto- and zooplankton and probably bacteria. In the water they appeared to be in a steady-state, renewed by biological activity and depleted by degradation and sedimentation. Particulate material should reflect this spectrum best, not only because phyto- and zooplankton themselves produce hydrocarbons and are part of the suspended material, but also because particles readily adsorb the dissolved but highly hydrophobic hydrocarbons. The authors' results support these assumptions quite well. The hydrocarbon spectrum of particulate material is thus characterized by: (I) a high content of n-alkanes belonging to a homologous series, which starts below n-dodecane, peaking with nheptadecane and with a pronounced uneven carbon number preference (CPI > 1); (2) a low content of higher molecular weight hydrocarbons with a CPI slightly greater than 1; (3) a predominance of HEH {three isomers); (4) a series of mono- and diolefins with uneven carbon numbers, most pronounced at C17, but also strong at nonacosane, and probably extended beyond Ca1 (the authors did not find, however, straight-chain polyolefins
65
with 19 carbon atoms); (5) a relativelylow concentration of the isoprenoid alkanes pristane and phytane, but a high concentration of squalene. The prevailing biological origin of the hydrocarbons in question is verified by the good agreement between the authors' findings, (1)--(5) above, and the data from the literature. A further support of their argument is the fact that their samples were taken from water containing a declining plankton bloom, which is evident from concentrations of nutrients, chlorophyll-a and D O C (A. Wenck, 1980, personal communication). The pattern of hydrocarbons remaining in the water from the plankton bloom is nearly as would be expected, according to literature data. This serves as a good proof for the relation between biological activityand hydrocarbon pattern. Most of the n-alkanes higher than tetracosane are probably derived from waxes of higher land plants (Eglinton and Hamilton, 1963; Kolattukudy and Walton, 1972). From the results of (2) above, it can be inferred that hydrocarbons coming from land run-off play a minor role as compared with the marine biogenic sources during a period of high biological activity,even in an area of high input of organic material by rivers. CONCLUSIONS
A correlation between particulate hydrocarbons and biological activity was already found by some authors. Goutx and Saliot (1980) found npentadecane and n-heptadecane predominating during plankton blooms in the Mediterranean; n-pentadecane partly made up 6 0 % of total hydrocarbons with concomitant high chlorophyll~ concentrations; at one station a significant correlation was observed between particulate n-alkanes and chlorophylls. A similar correlation between hydrocarbons and chlorophyll~ was stated by Zsolnay (1973) in an upwelling region off West Africa. The H E H seems to be a good indicator for phytoplankton activity.At the onset of a spring plankton bloom the concentrations of H E H in Bedford Basin increased sharply (Gordon et al., 1978), decreasing as the season progressed. The three diatoms Skeletonema eostatum, Detonula conferivacea and Thalassiosira nordenski~Idii, cultured in tank experiments, excreted mainly H E H during log phase growing, up to 0.6 -+0.13/~gmg -I dry weight of suspended material. After growth of the cultures had been stopped, concentrations dropped to zero within 12 weeks (Schulz and Quinn, 1977). Again, this demonstrates not only the relation between phytoplankton activityand concentration of H E H in suspended material, but also the replenishment of the highly unsaturated hydrocarbons in a natural population. The hydrocarbon pattern of dissolved or particulate matter is used as an indicator,whether or not this water is contaminated with petroleum hydrocarbons by oil or other anthropogenic sources, based upon the following premises: (1) A n uneven/even carbon number predominance, calculated for the range between n-dodecane and n-heneicosane, of CPI ~ 1 indicates anthro-
66
pogenic or geochemical origin; strong uneven carbon number predominance, CPI ¢ 1, indicates a biogenic origin of the hydrocarbon mixture. (2) A high concentration of n-alkanes in the range between ~-heneicosane and n-triacontane relative to the concentration in the range between n-dodecane and n-heneicosane as well as a high content of an unresolved complex mixture (UCM), strongly indicates crude oil or petroleum-derived hydrocarbons. (3) The absence or presence of aromatic and alicyclic hydrocarbons; the latter indicating pollution. (4) The phytane/pristane ratio as well as the ratios of pristane/n-heptadecane and phytane/n-octadecane; high ratios make pollution influence very likely. Comparing the authors' results with this set of conditions, the area of investigation may be classified as non-polluted in the above-mentioned sense. In addition they are evidence for biological activity unimpeded by pollution. Therefore, in order to estimate the degree of pollution by non-biogenic hydrocarbons and the effect of this pollution on life processes, the following two items should also be considered: (5) The content of HEH (and probably related highly unsaturated Cl9 and C21 hydrocarbons) and their relation to the sum of the other hydrocarbons. (6) The absence or presence of mono- and diolefins with carbon numbers, starting with C17 and extending b e y o n d C,9. To prove the applicability of this hypothesis, investigations on the seasonal dependence of these parameters should be continued. ACKNOWLEDGEMENTS
The authors thank Christian Wolff for recording and assistance in interpretation of the NMR-spectra and Manfred Ehrhardt for valuable discussions. This research was supported by grant No. MFU 0 5 0 6 / T V 1 of the Bundesministerium f~/r Forschung and Technologie.
67 APPENDIX List of algae species cited in literature to produce either HEH, n-Cls, n-C17, olefins and their occurrence in the Baltic Sea according to Pankow (1971 ) Class
Order
Variety/species
Occurrence
References*
Species
Variety only
Ulva lactuca Monostroma sp.
+ --
+ +
I, 5 1, 8
E n t e r o m o r p h a linza Enteromorpha compressa S p o n g a m o r p h a arcta H a l i m e d a sp. Halimeda discoidea Caulerpa sertularoides C o d i u m fragile Codium repens Codium cardioniarum Dunaliella tertiolata Prasiola stipitata Cystod~ctyon pavonium P l a t y m o n a s sp.
÷ @ --
+ q+
1 5 1, 8, 5
Benthic algae Clorophycea
Ulotrichales
Cladophorales Siphonales
Rhodophyceae
Volvocales Schizogoniales Siphonocladales Pyramimonadales Bangialea Cryptonemiales
Gigartinales
Rhodymeniales Ceramiales
Phaeophyceae
Fucales
Laminariales
Porphyra leucosticta D u m o n t i a inerassata Kallymenia perforata H a l y m e n i a sp. P e y s s o n n e l i a rubia Corallina o f f i c i n a l i s C h o n d r u s crispus Gracilaria e y l i n d r i c a Gracilaria b l o d g e t t i i Gracilaria m a m m i l a r i s Euchenma isoforme Euchenma compressa Rhodymenia palmata Rhody menia conferoides Ceramium rubrum P o l y s i p h o n i a ureeolata L a u r e n t i a corraUopsi~ L a u r e n t i a intricatu Ascophyllum nodosum F u c u s sp. Fucus distichus F u e u s spiralis F u c u s vesiculosus Sargassum sp. Agarum cribrosum Chorda filum Chorda tomentosa L a m i n a r i a digitata L a m i n a r i a saccarina L a m i n a r i a agardlin
--
--
6
---
---
6 6
--
--
10, 6, 5
---+ --
---+ --
6 6 3, 8 1 6
-+
+ +
10 I, 3, 5
+ --
+ --
5 6
--
+
6
-+ + -----+ -+ + -+
-+ + + + + --+ + + + --4-
6 4 5, 4 6 6 6 6 6
---
+ +
-+
+ +
--
+
-+ + + + --
-+ + + -b +
5, 4 1 5
5, 4 6 6
1, 8, 5, 4 4 1, 8, 5 5
1, 2, 8, 5 4 4 5 5
1, 5, 4 1, 8 5
68 APPENDIX (continued) Class
Order
Desmarestiales Ectoearpales
Punetariales Dictyotales Chordariales
Variety/species
Desmarestia viridis Ectocarpus fnsciculatus Ectocarpus siliculosus PilayeUa littorales Streblonerna oligosporum Scytosiphon lomentaria Punctaria latifolia Dictyota dichotoma Chordaria flagelliform is Leathesia difformis
()ccurrence
l~e ferenees*
Species
Variety only
~
~ ~
I , :~ I, i
4 ~
~ + ÷ ~-
10 I, 5 lO 1,8, 5
+
~
-+ ~
+ ~
5 6 5 5
÷ q-
+ +
3, 8
-
+ +
"
+
10 3, 8, 10 10 3, 8, 4
Planktonic algae Dinophyceae
Cryptophyceae
Euglenaphyceae Cyanophyceae Ptuinophyceae Desmophycea Cachoniaceae BaciUariophyceae (Diatomaphyceae)
Peridinales
Cryptomonadales
Euglenales Nostocales
Prorocentrales Rhizosoleniales
Coscinodiscales
Biddulphiales Naviculales Chrysophyceae
Xanthophyceae
Chrysomonadales
Chrysocapmdes Heterotrichales
Gongaulax polyedra Peridinium trochoideum Peridinum sociale G y m n o d i n i u m splendens Cryptomonns ovata Cryptomonas sp. R h o d o m o n a s lens Eutreptia viridis Euglena gracilis Synechoccus baciUaris Oscillatoria woronichinii Pyraminonns sp. Exuviella cassubica Cachonina niei Detonula conferivace Dity lum brightwellii Lauderia borealis Rhizosolenia setigera Thalassiosira fluviatilis Thalassiosira nordenski61dii S k e l e t o n e m a costatum Cyclotella nana Chaetoceros curvisetus Cy lindro theca fusiformis Navicula polliculosa Prymnesium parvum Syracosphera carterae Isochrysi~ galbana Coccolithus huxlegi Phaeocystis poncheti Tribonema aequale
d-
3, 8, 10
~
10
+
~
3, 8, 10
-
+ +
10 3 3
--
--
3
+
+
10
+
+
9
q+
+ +
3, 8, 10 3, 8, 10
+ + +
+ + +
3 3, 8 9
+
+
3, 8, 9, 1O, 7, 4
........
10
+
3
l-
+
+ Jr
+ + + -
3, 10 10
-t +
10
10 10, 4
+
3, 8, 10
~+ --
3, 8 3, 8 3, 8
* 1, Younllblood and Blumer (1973); 2, Halsall and Hill~ (1971); 3, Blumer et al. (1970); 4, Clark and Blumer (1967); 5, Youngblood et al. (1971); 6, Lytle et al. (1979); 7, Lee et al. (1970); 8, Blumer et al. (1971); 9, Schultz and Quinn (1977); 10, Lee and Loeblich (1971); 11, Pankow (1971).
69 REFERENCES Avigan, J. and Blumer, M., 1968. O n the origin of pristane in marine organisms. J. Lipochim. Res., 9: 350--352. Blumer, M., Mullin, M.M. and Thomas, D.W., 1964. Pristane in the marine environment. Helgol~inder wiss. Meeresunters., 10: 187--201. Blumer, M., Mullin, M.M. and Guillard, R.R.L., 1970. A polyunsaturated hydrocarbon (3, 6, 9, 12, 15, 18-heneicosahexaene) in the marine food web. Mar. Biol., 6: 226--235. Blumer, M., Guillard, R.R.L. and Chase, T., 1971. Hydrocarbons of marine phytoplankton. Mar. Biol., 8: 183--189. Burns, K.A. and Teal, M., 1973. Hydrocarbons in the pelagic Sargassum community. Deep-Sea Res., 20: 207--211. Cauwet, G., 1978. Organic chemistry of sea water particulates. Concepts and developmerits. Oceanol. Acta, 1: 99--105. Clark, R.C. and Blumer, M., 1967. Distribution of n-paraffins in marine organisms and sediment. Limnol. Oceanogr., 12: 79--87. Crisp, P.T., Brenner, S., Venkatesan, M.I., Ruth, E. and Kaplan, I.R., 1979. Organic chemical characterization of sediment-trap particulates from San Nicolas, Santa Barbara, Santa Monica, and San Pedro Basins, California. Geochim. Cosmochim. Acta, 43: 1791--1801. Derenbach, J., 1969. Partikul~re Substanz und Plankton an Hand chemischer und biologischer Daten, gemessen in den oberen Wasserschichten des Gotland-Tief im Mai 1968. Kiel. Meeresforsch., 25: 279--289. Eglinton, G. and Hamilton, R.J., 1963. The distribution of alkanes. In: T. Swain (Editor), Chemical Plant Taxonomy. Academic Press, New York, NY, pp. 187--217. Ehrhardt, M., 1969. The particulate organic carbon and nitrogen, and the dissolved organic carbon in the Gotland Deep in May 1968. Kiel. Meeresforsch., 25: 71--80. Ehrhardt, M., 1978. An automatic sampling buoy for the accumulation of dissolved and particulate organic material from seawater. Deep-Sea Res., 25: 119--126. Ehrhardt, M., Osterroht, C. and Petrick, G., 1980. Fatty-acid methyl esters dissolved in seawater and associated with suspended particulate material. Mar. Chem., 10: 67--76. Gearing, P.J., Gearing, J.N., Pruell, R.J., Wade, T.L. and Quinn, J.G., 1980. Partititioning of No. 2 Fuel Oil in controlled estuarine ecosystems. Sediments and suspended particulate matter. Environ. Sci. Technol., 14: 1129--1136. Gordon, D.C., Keizer, J.P.D. and Dale, J., 1978. Temporal variations and probable origins of hydrocarbons in the water column of Bedford Basin, Nova Scotia. Estuarine Coastal Mar. Sci., 7,243--256. Goutx, M. and Saliot, A., 1980. Relationship between dissolved and particulate fatty acids and hydrocarbons, chlorophyll-a and zooplankton biomass in Villefranche Bay/ Mediterranean. Mar. Chem., 8: 299--318. Halsall, T.G. and Hills, I.R., 1971. Isolation of Heneicosa-1, 6, 9, 12, 15, 18-hexaene and -1,6, 9,12, 15-pentaene from the alga Fucus vesiculosus. Chem. Commun., 448--449. Kolattukudy, P.E. and Walton, T.J., 1972. The biochemistry of plant cuticular lipids. In: R.T. Holman (Editor), Progress in the Chemistry of Fats and Other Lipids. Pergamon Press, Oxford, Vol. 13, pp. 121--175. Kremling, K., Osterroht, C. and Wenck, A., 1981. Investigations on dissolved copperorganic substances in Baltic waters. Mar. Chem., 10: 209--219. Lee, R.F. and Loeblich III, A.R., 1971. Distribution of 21:6 hydrocarbon and its relationship to 22:6 fatty acid in algae. Phytochemistry, 10: 593--602. Lee, R.F., Nevenzel, J.C., Paffenh~fer, G.A., Benson, A.A., Patton, S. and Kavannagh, T.E., 1970. A unique hexaene from a diatom (Skeletonema costatum). Biochim. Biophys. Acta, 202: 386--388. Lytle, T.F. and Lytle, J.S., 1979. Sediment hydrocarbons near an oil rig. Estuarine Coastal Mar. Sci., 9: 319--330.
70 Lytle, J.S., Lytle, T.F., Gearing, J.N. and Gearing, P.J., 1979. Hydrocarbons in benthic algae from the eastern Gulf of Mexico. Mar. Biol., 51 : 279--288. Morris, B.F., Butler, J.N., Sleeter T.D. and Cadwallader, J., 1977. Particulate hydrocarbon material in ocean waters. Rapp. P.V. Rdun. Cons. Int. Explor. Mer., 171: 107--116. Oro, J., Tornabene, T.G., Nooner, D.W. and Gelpi, E , 1967. Aliphatic hydrocarbons and fatty acids of some marine and freshwater microorganisms. J. Bacteriol. 93: 1811--1818. Pankow, H., 1971. Algenflora der Ostsee, Bd. I, II, III. Gustav Fischer Vertag, Stuttgart. Prahl, F.G. and Carpenter, R., 1979. The role of zooplankton fecal pellets in the sedimentation of polycyclic aromatic hydrocarbons in Dabob Bay, Washington. Geochim. Cosmochim. Acta, 43: 1959--1972. Schultz, D.M. and Quinn, J.G., 1977. Suspended material in Narragansett Bay: fatty acid and hydrocarbon composition. Org. Geochem., 1: 27--36. Simoneit, B.R.T., Mazurek, M.A. and Brenner, S., 1979. Organic geochemistry of recent sediments from Guaymas Basin, Gulf of California. Deep-Sea Res., 26A: 879--891. Thompson, S. and Eglinton, G., 1979. The presence of pollutant hydrocarbons in esturine epipelic diatom populations. II. Diatom Slimes. Estuarine Coastal Mar. Sci., 8: 75 --86. Wangersky, P.J., 1974. Particulate organic carbon: sampling variability. Limnol. Oceanogr., 19: 980--984. Wangersky, P.J., 1976. Particulate organic carbon in the Atlantic and Pacific Oceans. Deep-Sea Res., 23: 457--465. Wade, T.L. and Quinn, J.G., 1980. Incorporation, distribution and fate of saturated petroleum hydrocarbons in sediments from a controlled marine ecosystem. Mar. Environ. Res., 3: 15--33. Youngblood, W.W. and Blumer, M., 1973. Alkanes and alkenes in marine benthic algae. Mar. Biol., 21: 163--172. Youngblood, W.W., Blumer, M., Guillard, R.L. and Fiore, F., 1971. Saturated and unsaturated hydrocarbons in marine benthic algae. Mar. Biol., 8: 190--201. Zsolnay, A., 1971. A preliminary study of the dissolved hydrocarbons and hydrocarbons on particulate material in the Gotland Deep of the Baltic. Kiel. Meeresforsch., 27: 129--134. Zsolnay, A., 1973. Hydrocarbon and chlorophyll: correlation in the upwelling region off West Africa. Deep-Sea Res., 20: 923--925.