hydrocarbons of antarctic midwater organisms

Polar Biol (1986) 6:47 - 51

© Springer-Verlag 1986

Hydrocarbons of Antarctic Midwater Organisms

Susan B. Reinhardt and Edward S. Van Vleet

Department of Marine Science, University of South Florida, 140 Seventh Avenue South, St. Petersburg, FL 33701, USA

Received 23 April 1985; accepted 8 January 1986

Summary. H y d r o c a r b o n s have been i so la ted f rom mos t m a j o r b iomass species o f z o o p l a n k t o n and fish in an A n t a r c t i c mesope lag ic communi ty . Unl ike the odd-car - bon preference o f a l ipha t ic h y d r o c a r b o n s which typif ies te r res t r ia l p lan ts and t empera t e mar ine organ isms , even ca rbon cha in- length pa ra f f ins p r e d o m i n a t e in 80% of the An ta r c t i c species analyzed . A l t h o u g h the u l t ima te source o f these even c a r b o n n-a lkanes canno t be de te rmined f rom our s tudy, the d o m i n a n c e o f these c o m p o u n d s suggests an unusua l b iochemica l p a t h w a y m a y be respon- sible for their synthesis in this ecosys tem.

Introduction

Materials and Methods

Samples were collected in February of 1982 and during March and April of 1983 in Croker Passage located off the Palmer Peninsula (Fig. 1). Samples were collected using a 1.1 cm mesh, 5 m 2 mouth area, opening and closing Tucker trawl. Principal micronekton species from each trawl were identified, size-sorted, and kept frozen ( - 20 °C) until analyzed. All major biomass species of plankton and micronekton in this midwater community (as determined by previous trawls in 1982, T. Hopkins, personal communication) were analyzed. Further details such as weights extracted and number of organisms pooled can be found in Reinhardt (1984) or are available from the authors upon request.

In the laboratory, each sample was sonicated with 2:1 dichloro- methane: methanol, extracted overnight at room temperature, and filtered through a precombusted glass fiber filter. Internal standards (Sct-androstane and o-terphenyl) were added prior to extraction. Total lipids were fractionated into component classes by silica-gel chro-

In a previous s tudy o f l ipids in a three m e m b e r An ta rc t i c pelagic f o o d chain (the euphaus i id Euphausia crystallorophias, and the fish Pleuragramma antarcticum and Dissostichus mawsoni), analyses revealed an unusua l set o f even c a r b o n chain length h y d r o c a r b o n c o m p o u n d s in all three species ( N a c h m a n 1985). By cont ras t , in mos t te r res t r ia l p lants , ma r ine algae, and t rop ica l sponges and corals , the s t ra ight cha in h y d r o c a r b o n s exhibi t odd- c a r b o n p r e d o m i n a n c e (Egl in ton and H a m i l t o n 1965; Koons et al. 1965; C la rk and Blumer 1967; Whi t t l e et al. 1977; Sargent and Whi t t l e 1981). I f this unusua l hydro- ca rbon d i s t r ibu t ion is a pers is tent fea ture o f An ta rc t i c o rgan i sms , it no t only represents an unusua l b iochemica l p a t h w a y for these o rgan i sms , bu t m a y also p rov ide marke r s for t rac ing feeding re la t ionships in the An ta r c t i c f o o d web. W e have ex tended the studies o f N a c h m a n and Wi l l i ams to include samples f rom all m a j o r b iomass species o f z o o p l a n k t o n and fish ( a p p r o x i m a t e l y 20 species) in an A n t a r c t i c m i d w a t e r c o m m u n i t y in o rde r to see i f this unusua l h y d r o c a r b o n d i s t r ibu t ion is a sal ient fea ture o f the A n t a r c t i c ecosys tem.

~ o l ANTARCT,C

~'~ ~ PENINSULA LEIGE ISLAND ~ ° " ~ \ - - ~

GERLACHE STRAIT ( ,'~:c~

I ~ WEOO~L " \ % SEA /_ i ~

i AMERICA } ~1 ,) /

5' '8 60~/8 7 0 ' ~ 8 0 ~ II i ; ° 60'S \)5(~'S //

', ROSS '~) / /i/ \\ SEA ~ /

z \\

\ "-\

\ \ ANTARCTIC \\CONVERGENCE

Fig. 1. Antarctic midwater sampling site located in Croker Passage in the East Wind Drift system off the Antarctic Peninsula (* = area sampled)

40o8

48

matography (Nevenzel et al. 1965; Wakeham 1982; Reinhardt 1984). This procedure eluted: aliphatic hydrocarbons, aromatic hydrocarbons, wax esters plus steryl esters, triacylglycerols, and free fatty acids plus phospholipids with a series of solvents of increasing polarity. Only the hydrocarbon data is reported herein (other results can be found in Reinhardt and Van Vleet 1986). Aliphatic and aromatic hydrocarbon fractions were analyzed by gas-liquid chromatography using a Hewlett- Packard 5880A gas chromatograph equipped with dual flame ioniza- tion detectors. Hydrocarbons were analyzed on a 15 m x0.2 mm i.d. DB5 fused silica capillary column programmed from 9 0 - 2 5 5 ° C at 4°C min 1 (splitless injection, injection port temperature 240°C, detector temperature 250 °C, H 2 carrier gas 2 ml/min, H z make-up gas 27 ml/min). Peak identification was based upon comparison of reten- tion times to those of authentic standards and confirmation by com- bined high resolution gas chromatography/mass spectrometry with a Hewlett-Packard 5992B GC/MS system. Quantification was by peak area integration and comparison to internal standards after application of response factors determined for authentic standards. A procedural blank was processed with every three samples to assess the degree of contamination. Blanks were processed by carrying all solvents and internal standards through the entire procedure including column chromatography. Each column isolate for blank determination was analyz-

ed by gas chromatography as discussed above. All glassware was com- busted overnight at 500°C between sample batches to reduce contamination. Triplicate analyses of selected samples indicated a preci- sion of _+ 18°70 for the hydrocarbons and _+90/0 for total extractable lipids.

Results and Discussion

Total hydrocarbons generally constituted less than 1% of the total lipids in all species studied (Table 1), similar to levels reported for other marine organisms (Lee et al. 1971, 1972; Johnson and Calder 1973; Morris and Sargent 1973; Nevenzel and Menon 1980). Two types of hydrocarbons were analyzed: aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs). PAHs, possible indicators of,oil pollution or fossil fuel combus- tion products, have been isolated from Antarctic benthic organisms in concentrations ranging from 10 to 816 ng/g wet weight (Clarke and Law 1981). No measurable

Table 1. Hydrocarbons and total lipids of Antarctic midwater organisms collected in February 1982 and during March and April of 1983 in Croker Passage, Antarctica

Sample Depth Tli p 1 HC 2 Br 3 C: 04 C : 15 C" 26 Pris 7 O /E s CPI 9 collected (m)

Amphipoda Cyphocaris richardi Eurythenes 9ryllus Eusirus (perdentatus?) Paradania boecki Themisto 9audichaudi

Copepoda Calanoides acutus Metridia gerlachei Rhincalanus 9igas

Polychaeta Tomopteris carpenteri

Chaetognatha Sagitta oazellae

Coelenterata Atolla wyvillei Diphyes antarctica

Tunicata Salpa thompsoni

Mysidaceae An tarctomysis ohlinii

Euphausiacea Euphausia superba H 821° Euphausia superba Euphausia superba 4 . 0 - 4.6. cm Euphausia superba 3.4 - 3.6 cm Thysanoessa macrura

Vertebrata Electrona antarctica H 82 Electrona antarctica Electrona carlsbergi H 82 Pleuragramma antarcticum larvae Protomyctophum bolini H82

- 20.5 0.1 65.2 1.2 0.5 0.9 0.08 0.3 0.7 300-400 55.2 0.3 67.9 2.6 0.0 1.9 0.20 0.1 0.0 300 - 400 21.5 0.3 9.4 8.6 0.5 68.6 0.01 1.0 0.5 775 - 1000 19.4 0.1 65.8 7.2 0.5 1.5 0.05 1.0 1.3 130-200 18.5 BL . . . . 0.01 0.3 0.3

550- 820 45.0 4.0 73.2 0.1 0.1 0.7 2.93 1.5 0.7 750 - 820 21.4 BL . . . . 0.01 0.1 0.3 300-400 8.1 BL . . . . 0.01 0.4 0.0

230-480 8.4 0.4 1.7 15.6 0.5 - 0.01 0.7 0.7

550-820 17.4 0.2 3.5 29.4 6.3 - 0.01 0.1 0.2

630 - 820 1.1 0.2 51.0 14.3 1.3 1.2 0.11 0.3 ' 6.8 300 - 400 0.5 1.0 0.0 17.8 0.6 - 0.00 0.4 0.9

0 - 1000 24.3 0.1 9.4 62.5 1.7 14.5 0.01 1.8 3.4

550- 820 16.8 0.4 29.1 7.4 22.6 0.5 0.12 3.6 0.1

350-450 6.4 0.8 0.1 10.7 0.1 - 0.00 0.8 0.9 0 - 50 25.6 0.1 50.6 28.8 2.2 - 0.01 0.6 - 0 - 50 28.4 0.1 2.0 18.0 1.2 0.8 0.01 0.1 0.2 0 - 50 23.8 0.1 0.7 14.8 0.8 2.4 0.00 0.1 0.3

230-480 59.7 0.7 62.1 0.3 0.1 4.3 0.42 0.6 0.0

0 -1250 51.1 3.9 16.0 26.4 3.6 0.1 0.61 1.1 1.1 450 - 850 61.3 0.1 65.1 0.7 0.0 1.2 0.04 0.4 -

0 - 1 2 5 0 37.4 0.1 52.4 0.4 0.0 - 0.02 0.4 0.6 3 5 0 - 4 5 0 11.8 41.0 1.3 31.0 6.2 0.1 0.31 0.9 1.0

0 - 1 2 5 0 26.1 29.7 1.0 35.7 0.8 - 0.02 0.9 0.9

1 2 Tli p = total lipid (070 dry weight); THC = hydrocarbons (o7o Tllp); 3Br = Branched hydrocarbons (070 total HC); 4C : O = saturated hydrocarbons (o7o 7 8 total HC); 5C: 1 = monosaturated hydrocarbons (% total HC); 6C:2 = Phytadienes (% total HC); Pris = pristane (% Tlip); O / E = odd/even

hydrocarbons; 9CPI = carbon preference index; l°H 82 represents samples collected on 1982 cruise, all others are from 1983 cruise - = not detected

amounts of PAHs were detected in any of the midwater organisms analyzed in our study (detection lim- it = 2 ng/g wet weight).

Aliphatic hydrocarbons, measurable in all samples, were composed of branched hydrocarbons (pristane plus iso- and anteiso-compounds), saturated normal hydrocarbons, monounsaturated hydrocarbons, and phytadienes (Table 1). No polyunsaturates other than phytadienes were detected. Branched compounds were the major hydrocarbons (> 50% of the total hydrocarbons) in approximately half of the organisms studied. Pristane, generally the most abundant branched compound, was at least three times more concentrated (relative to total lipids) in the copepod Calanoides acutus than in any other species (Table 1). Pristane levels in Antarctic copepods were similar to reported values (0.01-2.94% of total lipid) for temperate species (Blumer et al. 1964). Concentrations in the temperature amphipod Themisto gaudichaudii (0.04%) and temperate chaetognath Sagitta elegans (0.05%) were also comparable to those in the Antarctic species analyzed (Blumer et al. 1964). Pristane levels in the krill Euphausia superba (0.01%) were lower than reported values (0.02-0.09%) for temperate eu- phausiids (Blumer et al. 1964), but the concentration in the Antarctic euphausiid Thysanoessa macrura (0.42%) exceeded this range. Analyses showed that T. macrura was feeding on the pristane-rich copepod C. acutus during the sampling period (T. L. Hopkins, personal communication), possibly explaining the enriched pristane levels in this euphausiid. C. acutus was not the major prey item for any other predator collected during this study.

In samples containing comparatively low levels of branched compounds, the most abundant hydrocarbons were straight chain aliphatics ranging from C14 to C39

with the most common n-alkane maximum at C16 or C21

(Fig. 2A). Seventy percent of the Antarctic species analyzed in this study had n-alkanes composed primarily of even carbon chain-length compounds, as indicated by odd-to-even ratios or less than one (Table 1). By contrast, most terrestrial plants, marine algae, and tropical sponges and corals, exhibit odd-carbon predominance in the straight chain hydrocarbons (Eglinton and Hamilton 1965; Koons et al. 1965; Clark and Blumer 1967; Whittle et al. 1977; Sargent and Whittle 1981). To eliminate bias caused by high concentrations of very short or long- chain compounds, the carbon preference index (CPI; Cooper and Bray 1963) was calculated for each Antarctic species. Eighty percent of the species analyzed had CPI values less than one, indicating an even-carbon predominance in the C20-C32 compounds. These results agree with the earlier study of Nachman (1985) who found even-chain compounds dominated the hydrocarbons of Euphausia crystallorophias, Pleuragramma antarcticum, and Dissostichus mawsoni from McMurdo Sound.

In contrast, Platt and Mackie (1982) reported hydrocarbon distributions in seven species of antarctic marine fauna, only one of which contained an even-carbon dom-

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uJ 0 Z <C 0

al

i-- z o

_1 w

A 3.4-3.6cm Euphausla superba THC=53 Fg/g DRY WEIGHT

30- O/E=O.1 CPI=O.3

20-

10-

~1'5 ' 1'7 '1'9 2'1 ! 2'3 ~ 25 ~' 2~7 r] ~9 r3 ;19 ;3 '3' ; 5 ~' 3~7 '3 '9 '

~ B Tomopterls carpenterl

THC=369 Fg/g DRY WEIGHT 15 O/E=0.7

CPI----0.7

10-

15-

115 r 17 119 2fl 2f3 ' 25 ' 2'9 1 ' 3'5 r 39 ' 41

C A R B O N CHAIN LENGTH

Fig. 2. Relative percent abundance and chain lengths of the normal hydrocarbons of two representative Antarctic mesopelagic organisms. Solid bars indicate mono-unsaturates and open bars indicate saturated compounds. THc indicates total hydrocarbons, O/E denotes total odd chain-length paraffins/total even chain-length n-alkanes, and CPI refers to carbon preference index

inance of n-alkanes. The only species analyzed in both studies was Euphausia superba. CPI's measured for this species in our study range from 0.2 to 0.9 (Table 1) while Platt and Mackie (1982) report a CPI of 1.1 for E. superba. Although Whittle et al. (1977) reported even carbon n-alkane dominance in a small number of marine organisms and tissues collected around the United King- dom (out of 255 samples), the dominance of these compounds in 70 - 80% of the species analyzed in the present study still represents a very unusual occurrence. Contam- ination by petroleum hydrocarbons was considered extremely unlikely in our study due to blank determina- tions showing negligible amounts of even or odd carbon- numbered alkanes and the aliphatic series in all samples being non-continuous from nCl4 to nC39 (Fig. 2).

In the present study, the percentage of organisms with even carbon predominance increased with depth. All deep dwelling organisms had O/E values less than one, while fewer mid-depth species showed this even- carbon predominance. The myctophid fish Electrona antarctica exhibited a year to year variation in hydrocarbon content with 1982 specimens having odd carbon predominance (O/E = 1.1) and 1983 fish, showing a preponderance of even chain-length paraffins (O/E = 0.4). This could indicate a dietary influence on the hydrocarbon stores with the even compounds origi- nating at lower levels of the food web. Concurrent gut analyses (T. Hopkins, personal communication) revealed

50

that 1982 E. antarctica were feeding upon the copepod Calanoides acutus (O/E = 1.5) while 1983 fish were filled with the krill Euphausia superba (O/E = 0.6), thus explaining the yearly variation in OEP for E. antarctica.

This study could not discern at which level in the food web the even predominance of hydrocarbons may have originated, since phytoplankton, nannoplankton, and bacteria were not collected. Of the five herbivorous zooplankton analyzed (the copepods Rhincalanus gigas, Metridia gerlache, and Calanoides acutus; the euphausiid Euphausia superba; and the tunicate Slape thompsoni), only two exhibited odd-carbon predominance (C. acutus and S. thompsoni). The diets of these herbivores have not been sufficiently studied to allow identification of phytoplankton prey species. These herbivores may be eating several species with varying hydrocarbon com- positions or they may be producing the even carbon- chain compounds themselves.

Even carbon predominance in 80% of the Antarctic midwater species suggests the utilization of unusual biochemical synthetic pathways in this food web. One mech- anism proposed for n-alkane synthesis is decarboxylation of fatty acids (Ackman and Hansen 1967; Han et al. 1969). If this metabolic pathway is being used, relative- ly high concentrations of odd-numbered fatty acids (O/E > 1) should have been present in all organisms with hydrocarbon CPIs less than one. This was not observed (Table 2), indicating that another pathway was operat- ing. Hydrocarbons can be synthesized in zooplankton by alcohol reduction (Ackman and Hansen 1967; Clark and Blumer 1967; Avigan and Blumer 1968), however the major alcohol components of wax esters (as determined by GC/MS) in the Antarctic organisms were 14: 0, 17 • 1, 19 : 1, and 21 : 1 (Reinhardt and Van Vleet 1986), in contrast to the major hydrocarbons (16:0 and 21"0), indicating that reduction of alcohols was not the primary

Table 2. Odd-even ratios of hydrocarbons and fatty acids in selected Antarctic mesopelagic organisms

Sample HCs a FAMEs b Odd/Even Odd/Even

Wax esters Rhincalanus gigas 0.37 0.79 Tomopteris carpenteri 0.67 0.04 Atolla wyvillei 0.27 0.02

Triacylglycerols Calanoides aeutus (Group 1) 1.45 0.08 Metridia gerlaehei (Group 2) 0.14 0.22 Rhincalanus gigas (Group 3) 0.37 0.40

Phospholipids Rhincalanus gigas 0.37 0.37 Cyphocaris richardi 0.31 0.07 Metridia gerlachei 0.14 0.45 Calanoides acutus 1.45 0.01 Atolla wyvillei 0.27 0.04 Pleuragramma larvae H82 0.94 0.02 Electrona antarctica viscera 0.27 0.01

a HC = Hydrocarbon; b FAME = Fatty acid methyl ester

biosynthetic pathway for synthesis of even-chain hydrocarbons. The true biosynthetic pathway for these hydrocarbons is presently unknown.

Even carbon-chain hydrocarbon predominance is an important feature of the lipids of Antarctic midwater fish and macrozooplankton in the Croker Passage, oc- curring in 80% of the major species of the community sampled. From our study, we cannot determine whether these compounds are synthesized de novo by many members of this food web, or are synthesized by a few key members and passed along the food chains. What is clear, however, is that the ultimate source of these even carbon n-alkanes requires a rather unusual biochemical pathway for their synthesis. The significance of these unusual compounds in this Antarctic ecosystem remains unclear at the present time.

Acknowledgements. We would like to thank T. L. Hopkins and B. H. Robison for collecting this unique sample set, identifying the species, analyzing gut contents, and supplying additional data necessary for this research; and J. J. Tortes, W. C. Reinhardt, and H. L. Baker Jr. for their editorial assistance. Support was provided by NSF grant DPP-80- 23961. Sampling support was provided by NSF grants DPP 81-07510 and DPP-81-19621 to T. L. Hopkins and B. H. Robison.

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Avigan J, Blumer M (1968) On the origin of pristane in marine organisms. J Lipid Res 9:350-352

Blumer M, Mullin MM, Thomas DW (1964) Pristane in the marine environment. Helgol Wiss Meeresunters 10:187-201

Clark RC Jr, Blumer M (1967) Distribution of n-paraffins in marine organisms and sediment. Limnol Oceanogr 12:79-87

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Eglinton G, Hamilton RJ (1965) The distribution of alkanes. In: Swain T (ed) Chemical plant taxonomy. Academic Press, New York

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Nevenzel JC, Rodegker W, Mead JF (1965) The lipids of Ruvettus pre- tiosus muscle and liver. Biochemistry 4:1589-1594

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hydrocarbons of antarctic midwater organisms

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