Steroid geochemistry in the oxygen minimum zone of the eastern tropical North Pacific Ocean

0016-7037/87153 00 + .oo

Geoehimua d CosmochrmzcaActa Vol. 5 I, pp. 305 I-3069 0 Palplmoa Journals Lad. 1987. Rinted in U.S.A.

Steroid geochemistry in the oxygen minimum zone of the eastern tropical North Pacific Ocean STUART G. WAKEHAM* Department of Chemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, U.S.A. (Received April 23, 1987; accepted in revisedform August 25. 1987) Abstract-Particulate matter was collected in the eastern tropical North Pacific Ocean, an area characterized by a shallow and intense oxygen minimum zone, in order to investigate the geochemistry of particulate organic matter in the ocean. Sinking, large particles and suspended, small particles were analyzed for sterols, 3-keto-steroids, 3-methoxy-steroids, and sterenes. Vertical tluxes of steroid classes in sinking particles and concentrations of steroids in suspended particles decrea& rapidly below the euphotic zone consistent with upper ocean sources and deep water decomposition. Significant compositional changes were observed with increasing depth and as a function of particle size. Sinking particles were enriched in C2,-sterols but deficient in C&- and C&sterols compared to the suspended particles. Suspended particles, especially in the oxygen minimum zone, were enriched in steroidal ketones and stones compared to sinking particles. Steroid distributions suggest that the oxygen minimum zone is the site of active “diagenetic” transformations, most likely microbially-mediated, of stenols to steroidal ketones, stanols, and sterenes. These transformations occur preferentially in suspended particles relative to sinking particles. INTRODUCTION PARTICULATE ORGANIC MATTER in the oceanic water

column plays an important role in carbon cycling. Particles provide nutrition for heterotrophic organisms throughout the water column, their decomposition releases nutrients into the water column for more biological production, and their sedimentation transports material to the sea floor. The nature of particles depends on a balance between the biological processes of production and consumption and the physical processes of aggregation, disaggn&on and transport. Two classes of particles are generally recognized on the basis of sampling techniques, although a continuum of sizes exists in the water column (MCCAVE, 1984). w, sinking macrwtes are quantitatively rare, but their high sinking velocities and short turnover times make them the dominant mechanism for transporting material, including labile organic compounds, to the sea floor. Sediment traps are thought to collect mainly large, sinking particles. Small, suspended particles, on the other hand, are characterized by very low sinking velocities and long turnover times. However, the quantitative abundance of suspended particles means they constitute most of the standing stock of particles in the water column. Suspended particles are mainly sampled by filtration. The organic chemical composition of particulate matter in the ocean has been investigated over the past decade (see review by LEE and WAKEHAM, 1988). One class of organic compounds which has received substantial attention is the steroids, particularly because the diverse structures of biogenic steroids and their transformation products are of great use in elucidating source and organic matter alteration processes which

l Present address: SkidawayInstitute of Oceanography, P.O. Box 13687, Savannah, GA 3 1416, U.S.A.

may be involved in the generation of petroleum. The relatively stable steroid hydrocarbon skeleton can incorporate a variety of functional groups, including hydroxyl and carbonyl groups, and olefinic linkages in both the steroid nucleus and various alkyl side-chains at C- 17, yielding classes of compounds which may be used to trace reaction mechanisms and rates. As a result, the biochemistry of steroids and their distributions in marine and terrestrial organisms have been extensively investigated (reviewed by NES and MCKEAN, 1977; GOAD, 1978; VOLKMAN, 1986; and as cited below). Biochemical and diagenetic transformations of steroids in Recent and immature sediments have also been examined (e.g. GAGOSIAN et al., 1980; MACKENZIEet al., 1982; BRASSELLand EGLINTON, 1983; and references cited therein and below). The oceanic water column represents the transition zone between surface water and continental sources of organic compounds and the sink for material at the sediment-water interface. Most of the particulate organic matter in the water column is produced by marine organisms inhabiting surface waters; except for coastal areas, terrestrial inputs are smaller and remain poorly quantified. Sediment trap experiments have shown that most organic compounds associated with sinking particles are transformed or decomposed as the particles sink (LEE and WAKEHAM, 1988). The organic geochemistry of suspended particles has received less attention. Aspects of particulate steroid biogeochemistry have been discussed previously (GAGOSIAN and NIGRELLI, 1979; GAGOSIANand HEINZER, 1979; SALIOT et al., 1982; GAGOSIAN et al., 1982, 1983; WAKEHAMet al., 1984a,b). To date there has not been a systematic comparison of steroid geochemistry in both small and large particles collected at the same location and including the major precursors and transformation products involved in steroid alterations as described for sediments.

3051

S. Cl. Wakeham

3052

7

-22

T*C s %. 0

---6 . .....33.5

24 (2 340

26 18 345

28 24 350

30 30 355

0 0

0.6 150

08 200

1.0 250

Plqmcntr pq/l 02 pM a9 -

- -

__--* , I \ I

300 -

I I-P19ments

I z

04 100

02 50

600 -

I

:

I I t x

:

I ,

900 -

I.

I I

IT ,

1200 -

s

Qt

I 1500 -

2

’

FIG. 1.Hydrographic conditions at the VERTEX II/III site (BROENKOW and KRENZ,1982; BROENKOW et al., 1983a, b). T = temperature, S = salinity, u, = density, Or = dissolved oxygen, pigments = fluorescing material.

The VERTEX (Vertical Transport and Exchange) program was initiated to characterize the production and exchange of material in the upper 2000 m of the ocean. One component of this coordinated effort was an investigation of the organic geochemistry of particulate matter in the water column. During the VERTEX II and III cruises to the intense oxygen minimum in the eastern tropical North Pacific Ocean off western Mexico, sinking particles and suspended particles were simultaneously collected in sediment traps and by in situ filtration, respectively. Sterols (stenols and stanols), steroidal ketones (stenones and stanones, or collectively sterones), and steroidal hydrocarbons (sterenes) were determined in these samples (along with other lipids; e.g. WAKEHAM and CANUEL, 1987). This paper describes steroid distributions in the VERTEX II and III particles and the implications of these observations on steroid geochemistry in the ocean.

METHODS

that live zooplankton (“swimmers”) were caught in some traps especially those above the oxygen minimum. Inclusion of swimmers in the mesopelagic traps was less likely. Nevertheless, the use of HgC12as a preservative precluded removal of swimmers, so steroid data from the epipelagic traps must be. viewed with this in mind. Suspended particles were sampkd using WHISPs (Woods Hole in situ pumps). These pumps are hydrographic-wire mounted stream-powered large-volume filtering systems. Typically, 3OO-4000 1 of seawater (Tabk 1) were pumped through ashed-glass fiber filters (293 mm, type A/E, nominal pore size 1 pm) during 5-14 hr deployments at night. A Soutar box core was collected in 3500 m of water during VERTEX III. All samples were stored frozen until analysis.

Analysis Sediment trap samples were lyophylized and extracted with methylene chloride. WHISP samples were Soxhlet extracted with toluene-methanol( 1:1).(The use of two extraction protocols resulted from the need by other VERTEX collaborators

Table 1.

Deploywnt data for WISPS and PITS during VERTEX II and III.

Sampling The VERTEX II and III cruises of R/V Wecoma sampled the oxygen minimum xone in the eastern tropical North Pacific about400kmoffManzanillo,Mexiw(15-18”N, 107-109°W) during October-November 1981, and November 1982, respectively. This region is characterized by an intense oxygen minimum extending between 100 and 800 m depth (Fig. l), with minimum diwlved oxygen concentrations of less than about 1 PM kg-’ between 110 and 250 m (BROENKOW and KRENZ,1982; BROENKOW et al., 1983a). Mean primary productivity rates of 860 and 470 mg C m-* d-* were measured during the cruises in 198 I and 1982, respectively (KNAUER et al., 1984), indicating the area to be mesotrophic. Fluorescence (pigment) profiles showed the biomass maximum to lie at about 60 m depth. Secondary and tertiary fluorescence peaks at 120 and 400 m (BROENKOW et al.. 1983b) were consistent with “hot spots” (KARL and KNAUER,1984) of bacterial activity. Sinking particles were collected (Table 1) in free-floating Soutar-type particle interceptor traps (PIT$O.25 m* collecting area with l-cm grid at the top of the cone; MARnN et al., 1983). Mercuric chloride was used to minimize sample deterioration during deployment of the traps. Deployment periods were 21 d in 198 1 and 19 d in 1982. It was inevitable

YHISPs

“;$h

Date

Filtering time (h) VERTEX

5 200 1000

11/14/81 11/12 11/14

1:: 450 950 1450

11/20/82 11127 11/22 11126 11126 11/23

1734 1912 2576

::

Depth

1: 470 947 1500

III

: 9.5 10 :8

Volume flltered (1)

II

5

VERTEX

5

PITsI

1053 3z 1727 1024 4062

100 450 1500

1VERTEX II PITS deployed 28 October 18 Novellber,1981; VERTEX III PITS deployed 10 Novelaber- 29 November, 1982.

3053

North Pacific steroid geochemistry to analyze the sediment trap material for trace levels of chlorinated hydrocarbons. Their extraction scheme required the use of pesticidm methylene chloride, and ahquots of this exttact were provided for lipid analysis.) Lipids were fractionated by adsorption chromatography on silica gel (Merck 60,5% deactivated with water) into constituent classes on the basis of their polarity. Hydrocarbons (including stemnea) were eluted with hexane, nuclear saturated 3-keto-steroids (stanones) and 3-methoxy steroids (steroid methyl ethers) were eluted with 10% ethyl acetate in hexane, nuclear unsaturated 3-ket+steroids (A’-stenones) and Cmethylsterols with 15% ethyl acetate in hexane, and 4desmethylsterols (both stenols and stanols) with 20% ethyl acetate in hexane. The classes were analyzed by capillary gas chromatography (sterols as acetates and TMS-ethers) using a Carlo Erba Fractovap 4160 with on column injector and a 25 m X 0.3 mm i.d. glass capillary column coated with a 0. I5 cm film of immobilized SE-52 or a 30 m X 0.25 mm i.d. fused silica capillary coated with a 0.25 pm him of DB5 (methyl silicone gum). Hydrogen was used as carrier gas. Concentrations were calculated relative to internal standards added to samples before gas chromatography (androstane for hydrocarbons; cholestane for steroidal ketones and alcohols). Gas chromatography-mass spectmmetry analyses were performed on a system combining a Carlo Erba 4 160 gas chromatograph, a Finn&an 4500 mass spectrometer, and an Incas 2300 data system. Structural as-

Teble 2.

Degh

signments were made on the basis of coinjections and mass spectra of authentic compounds when available, by comparisons with literature spectra (e.g. LEE et al.. 1979; GAGOSIAN and HEINZER, 1979; WARDROPER, 1979; SMITH, 1984; and references therein), or by mass spectral interpretation.

RESULTS Vertical fluxes and concentrations Fluxes of steroid classes associated with sinking particles collected in the PITS and concentrations of steroids in suspended particles sampled by the WHISPs are given in Table 2. Higher vertical fluxes for steroids and particulate organic carbon (POC) out of the euphotic zone (e.g. 100 m PITS) for VERTEX II compared to VERTEX III may refkct the 2-fold higher primary productivity in 198 1 than in 1982. POC fluxes in 1981 were about 2.5X higher than in 1982, consistent with the productivities and showing reasonable primary productivity-POC flux coupling. However, fluxes for the various steroid classes were less well coupled with primary productivity; ratios of 198 l/1982

Vertlcel fluxes of sterolds and particulate organic carbon (POC) In slnklng parttcles and concentrations In suspended particles.

4-DesmeWlsterolr

cuetwlsterols

Steoones

Stenones

Sterotd ethers

Stennes

VERTEX 11 PITS h&s2

:K

88::

100 140 29

A:; 0.5

::;

d)

37.8 26.6

25.; 3:6

f:2' 3.

:::

X:2'

::: 0.8

::: 0.3

VERTEX III PITS

::

eo

::t 0.4

f:f 1.5

::: 0.6

1.5 ::(:

0”:: 0.1

VERTEXII WISPS ln911) 180 E

4.6 X:'5

1:::

33.4

10.6

2.3

17.6 5.1

:::

0.8 0"::

VERTEX I11 WISPS (n90) 162 220 :;

::: 0.5 1.5

6.6 9.3 2.2 5.3

1:

::;

::o"

29.9 ::: 0.9

1::: 12.9 0.4

9.7 2.2 11.5

;:;

1.3 2.0

0.9 f::

0.6

0.5

Sedirnt Floe (UOl9) 3.9

0.3

0.4

1.3

76

S. G. Wakeham

3054

steroid flux at 100 m varied between 3.6 for 4-tnethylsterols and stenones to 16 for sterenes. However, despite order of magnitude differences in flux out of the euphotic zone for the two years, fluxes at 1500 m were similar. Thus, these data suggest that two-fold variations in surface water primary p~u~vi~ rates can lead to substantial variations in steroid flux in the upper ocean, but to relatively small variations at depths of 1500 m or more. In contrast to the marked variation in steroid vertical flux between 198 1 and 1982 and regardless of the difference in primary productivities, concentrations of steroids associated with suspended particles were remarkably similar both years. For the more detailed depth prolile of VERTEX III, concentration maxima for all classes except sterenes were observed at 60 m, the depth of the biomass maximum; sterene concentrations were highest in the oxygen minimum zone. Weight ratios of steroid class concentrations to POC concentrations (Table 3) indicate qualitative differences between the sinking and suspended particle classes. Decreased steroi&POC ratios suggest preferential degradation of the steroidal compounds while increased ratios indicate either preferential preservation or perhaps even in situ production. Thus, for example, 4desmethylsteroi:POC ratios generally decreased with increasing depth, consistent with the dominant sterol sou~x: being in the euphotic zone and with labile sterols being preferentially degraded relative to total carbon with depth. On the other hand, increased sterene:POC ratios measured in particles from the oxygen mi~mum zone are indicative of in situ production ofsterenes as particles pass through oxygen-depleted waters.

I-llesmethyl- and 4-rn~~y~stero~~ Distributions of sterols in sinking (PIT) and suspended (WHISP) particles from the two VERTEX

VERTEX If PITS 60

47.0

0.21

1'2

f?: 35:9 12.6

o"% 0:lB 0.22

100 450 15no

19.0 48.2 38.1

0.24 0.26 0.19

5

4.3

0.11 0.16 0.05

:;

:::5 1.02 0.56 1.6

0.67 1':; 0.82 1.4

:% oh1 0.23 0.35

0.02 0.13 0.17 0.06 0.13

0.27 0.57 0.29

% 0114

0.02 0.02 0.05

EZ 0.46

0.25 0.10 0.12

0.02 0.30 0.05

0.17

0.02 0.03

WITEX III PITS 0.62 0.70 0.71 VERTEX ii YWPS

lE

:::

0.14 0.61 0.21

VERTEX 111 YHISPS 60" :: 1%

i::

0.19 0.13 0.03 0.14 0.05 0.08

0.05

om4

::: :::

0.16 0.15 0.12 0.46 0.14 0.25

0.71 0.54 ::& 0.15 0.14

E: 0:or 0.12 0.17

0":: 0.05 0.08

0‘02

0.008

o.[to7

Sadbent FloC 0.005

cruises are shown in Figs. 2 and 3. A’- and A’*“- Stenols dominated, followed by As2*28)-stenols in many cases; A’-stenols were generally not abundant. The major sterol in all samples was cholest-S-en-38-01 (5. in Figs. 2 and 3 and in Table 4). Cholesta-5,22Edien-38-01 (3X another C27 sterol, was also an impo~nt constituent. Phytosterols were dominated by the C2, compounds 24-methylcholesta-5,22E-dien-3&ol (7) and 24-methylcholesta-5,24(28)_dien-30-01 (9). Small amounts of 23,2~imethyIchol~~-5,22E~en-3~~1 (II). 23,24_dimethylcholest-5-en-38-01 (14), and 24ethylcholesta-5,24(28)-dien-3j3-ol (17) were also present. C29sterols, primarily 24-etbylcholesta-5,22Edien3&01(12f and 24-ethylchol~t-5~n-3~-ol (unspeciiied stereochemistry but possibly /?-sitosterol as discussed below, l5) were surprisingly abundant, especially in the WHISP samples. The sediment floe (Fig. 2) was dominated by cholest-5en-3&ol and 24_ethylcholest5-en-38-01. Sa-Stanols were much less abundant than stenols in all samples, but stanol:stenol ratios varied significantly with both depth and particle size (see discussion below). 4-Methylsterols were present, but they were minor components in all samples. In the PITs, 4-methyl sterols were 0.5-2% of total sterols. In the WHISPs, however, they constituted up to 9% of the sterols and were 10% of the sterols in the sediment floe. 4,23,24-T& methylcholest-22E-en-3/3-ol(18) was the only Cmethylsterol present in significant quantities, usually being I-4% of total sterols and up to 90% of the 4-methylsterols. Other less abundant ~methyl~erois, as determined by mass spectrometry, included 4-methyl-24ethylcholest-24(28)-en-36-01 (about 5% of 4-methylsterols in all samples), 4,4-dimethylcholestan-313-01 (2%), ~methyl-24~thylchol~~n-3~~1 (2%), 4-methylcholestan_3j??-ol ( 1%) and 4-methyl-24-methylcholestan-38-01 ( 1%). The overall distributions of sterois in the two particle classes were generally similar, but significant and consistent differences related to particle size were apparent. The relative abundance of cholest-5-en-38-01 was consistently and substantially greater in sinking particles (34-62% of total 4desmethyl sterols in VERTEX II and 53-8 1%in VERTEX III; Fig. 4) than in suspended particles ( 17-39%). Suspended particles in the oxygen minimum were conspicuously depleted in cholest-5en-3&ot. Phytosterols were usually relatively more abundant in suspended particles than in sinking particles; for example, 24-methylcholesta-5,22E-dien-3& 01 was 13-20% of total sterols in the WHISP samples compared to 5- 17% in PIT samples (Fig. 4). Similarly, suspended particles were considerably enriched in C2, sterols (e.g. 24-ethylcholest-5-en_38-01, Fig. 4) relative to sinking particles. Suspended particles were enriched in S~Z-stanols compared to stenols. Total Sa-stanols in sinking and suspended particles in the euphotic zone were 4-6% of total sterols. For sinking particles, there was little change in stanokstenol ratios with increased depth. However, stanols became substantially more abundant in suspended particles in the oxygen mini-

RELATIVE PERCENT

S. G. Wakeham

3056

VERTEX III WHISPs 3’o 119

12

A0

6

CIA5

0

2 4 6 8 IO 12 14 i6 18

29l-l

II a17

PITS

mA2*

q A5.22 A5.24(281

2 4 6 8(0

12 WI618

6 w SL”

2468K)l21411il6

6

0

24681012141618

2 4 6

8fO1214b#l

2466tO12141618

FIG. 3. Distributions of sterols in the VERTEX III PIT and WHISP samples. Designations refer to Table 4.

3057

North pacific steroid geochemistry Teble 4. Sterols lssigned In VERTEX 11 end III pertlcles.

DesIgnWon (Figs. 2 end 3) 1 : 4 : I: 1: ;: ;: 15 ;; 18 19

Assigment 24-norcholeste-5,22E-dlm-)l-ol 27-nor-50athylcholcsto-5.22E-dlen-3~-ol choleste-5,22E-dlen-lo-01 5~cholest-22E-en-3~01 cholcst-&en-L-o1 5~choleston-30-01 24-rthylcholeste-5,22E-dien-3s-ol 24-mthy1-5~Aolest-22E-en-34-ol 24-rthylcholesto-5.24~28I-dien-3~-ol 24-methylcholcst-5-e~36-01 23.24-dl~t~lcholcste-5,22E-dlen-3r-o1 24-ethylcholestr-5,22E-diem30-01 24-ethyl-So-cholcst-226en-36-01 23.24-dimethylcholcst-5-en-3e.-ol 24-cthylcholest-kn-3wol tl-ethylcholestm-38-01 24-ethylcholeste-5,24(28)-dien-36-01 4.23,24-trlrthylcholest-22-en-3,-01 4.23.24-trlrthylcholestdn-3s-ol

change greatly with depth. Suspended particles, on the other hand, did show some notable depth-related compositional changes. Stanones dominated the euphotic zone samples, the major constitutents being 5& and Socholestan-3-one @and 5J and 23,24-dimethyl5&cholestan-3-one (14); stenones were relatively minor. As depth increased, the abundance of 23,24-dimethyl-5&cholestan-3-one (14) decmased markedly while A’-stenones (chole&-en-3-one (3J, cholesta4,22dien-3-one (lJ, and 24methylcholesta4,22dien3-one (6J) increased in importance. For VERTEX III, A4-stenones dominated the steroid ketone distribution in the PIT samples and in the WHISP samples from 450 m and deeper. The two deepest VERTEX III WHISP samples (950 and 1500 mj alsocontained 4,23,24-trimethylcholest-22-en-3-one (dinosterone, 15)

VERTEX II VERTEX IU awbst-5-en-3/?-ol /XI 20 40 60 20 40 60 80 nents) at the apices (Fig. 5). For example, the sinking particles consistently contained higher abundances of Cz, sterols than the suspended particles, while suspended particles were enriched in Cz9 sterols. Moreover, the carbon-number distributions indicate that suspended particles fall into three distinct depth-related groupings: 1) particles from the euphotic zone (5-60 m); 2) particles from within the oxygen minimum zone (140-470 m); and 3) particles collected below the Orminimum (950-1500 m): this separation is less clear for the sinking particles in these depth intervals. The sediment sample plots nearest to the suspended particles from below the O*-minimum.

__

Steroidalketones A’-Sten-3-ones and nuclear saturated stan3-ones in sinking and suspended particles are reported in Figs. 6 and 7. Vertical fluxes and suspended particle concentrations of 3-keto-steroids decreased with depth below the euphotic zone (Table. 2). Stenones were more abundant than stanones in the shallowest and deepest PIT samples; stanones tended to be more abundant at intermediate depths. However, in the WHISP samples, stanones were considerably more abundant than stenones in the euphotic zone and in the upper part of the oxygen minimum zone, below which stanone and stenone abundances varied but were roughly equivalent. Compared with sterols, steroidal ketones were significantly enriched in the suspended particles (steroidal ketone:sterol ratios were between 0.03-0.23 in PIT samples vs. 0.20-0.72 for WHISP samples). Steroidal ketone distributions in sinking particulate matter for the two years were generally similar. Major stenones were cholest4en-3-one (2 in Figs. 6 and 7 and in Table 5), cholesta-4,22dien-3-one (lJ, and 24 methylcholest&,22dien-3-one (6J 5B- and Sor-cholestan-3-one @ and 5J and 24methyl-5&cholestan-3one (9J were abundant among the stanones. The steroidal ketone composition of sinking particles did not

~-Et~k~olost-S-~-3~-ol

0

20

0

I%’ 20

FIG. 4. Vertical distributions (percent of total sterols) for cholest-S-en-3&ol,24-methykhoksta-S,22dien-3/9-ol,and 24 ethylcholest-5cn-3& for VERTEX II/III PIT, WHISP, and sediment floe samples (3500 m).

S. G. Wakeham

3058

PtTs

WHISP 0 0 b

PIT s w&tic D %-min

3 VERTEX II 5 VERTEX IU

s belowC&-min

S Sediment

FIG. 5. Ternary diagrams of Cz7vs. CB VS.CB compounds for sterols, steroidal ketones, and sterenes in VERTEX II/III PITS and WHISPs. Ail PIT and WHISP samples are indicated in each diagram. PIT depths and contours are assigned in the feft set of diagrams, WHISP depths and contours in the right set. U~erlin~ depths (5) are VERTEX II, non-underling depths (5) are VERTEX III.

as the second most abundant component. This stanone was only a minor constituent of all other particle samples. The sediment floe contained 4x more stanones than stenones, and 24-methylcholest-22-n-3-one (1) was the major component. Plotting sterones in a triangular diagram (Fig. 5) did not reveal the sharp distinctions observed for the sterob; PIT and WHISP samples were more closely related. However, there is a suggestion that PIT particles in the Or-minimum might be somewhat enriched in Cr9 components compared to shallower and deeper samples. In contrast, it is the suspended particles from below the oxygen minimum which seem to contain the greatest abundances of Cm ketones. The sediment floe contained the lowest proportion of Cz8compounds. Steroidal methyl ethers Small amounts of fmethoxy-steroids were detected in fractions also containing stanones. Detailed distri-

butions of methyl ethers were not dete~in~, however, the four major components were 3-methoxycholesta5,22-diene, 3-methoxycholest-5-ene, 3-methoxycholestane, and 3-methoxy-24methylcholesu+5,22diene, 3-Methoxycholest-Sene and Emethoxycholesta-5,22diene were. respectively, the two most abundant ethers in all samples. Vertical fluxes of steroidal methyl ethers and concentrations of steroidal methyl ethers associated with suspended particles (Table 2) were greatest in the region of the biomass maximum, below which they decreased with increasing depth. PIT samples contained steroidal ethers at levels of S- 17% of the sterols. Sterenes Steroidal hydrocarbons were particularly conspicuous in suspended particles where they were considerably more abundant than the n-alkanes of similar carbon numbers, for example sterenes constituted 88% of total acyclic + cyclic hydrocarbons in the 140 m

f

I

.

1

RELATIVE

PERCENT

3060

S. G. Wakeham

VERTEX III WHlSPs 12

q 5flA”

6

5aA”

0

Cl

iH A22

12

PITS a

A4

n A422

6

22

0

2

Y i=

4 6

6 10 12 14

2

4 6

6

2 4

am

8 10 12 14

45Om

6 8 101214

022 _

6

2 4 6 8 10 12 14

6

2

12

12

6

6

0

0

FIG. 7. Distributions of steroidal ketones in VERTEX to Table 5.

4 6 8 101214

2 4 6 8 10 12 14 III PIT and WHISP samples. Designations refer

306 1

North Pacific steroid geochemistry Table 5.

Steroid ketones asslgned In VERTEX particle samples.

Designation (Figs. 6 and 7) 1 2 3 2 7 : !Y 12 13 14 15

Asslgment cholesta-4.2%dien-J-one cholest-22-en-3-one cholest-4-en-3-one 5a-cholestan-3-one 5a-cholestan-3-one 24-methylcholesta-4,22-dien-3-one 24-methyl-5.-cholest-ZZ-en-3-one 24-methylcholest-4-en-3-one 24-methyl-5e-cholestan-J-one 24-ethylcholest-4-en-3-one 23.24-dinlthyl-5s-cholestan-3-one 24-ethyl-5e-cholestan-3-one 24-ethyl-5a-cholestan-3-one 23.24-dimethyl-5~cholestan-3-one 4.23.24-trirthylcholest-22-en-3-one

sterenes constituted 50-8596 of the PIT sterenes, vs. 50-70% for WHISP samples. It should be noted that the extreme range of CZ7compounds in the O2 minimum PIT samples reflects the fact that VERTEX II samples contained substantiaUy more Cz7 sterenes than did the VERTEX III samples. Cz9 sterenes accounted for only 3-25% of sterenes in both PITS and WHISPs. The relative depletion of Czs sterenes in suspended particles in the O2 minimum contrasts sharply with the abundance of Cz9 sterols in the same samples. Within a given carbon number, steradienes and steratrienes were more abundant than corresponding monosterenes. For example, the ratio of (C2, + C2, + C&-monoenes:dienes:trienes in the VERTEX III WHISP samples was 1.0~2.8:1.8 at 60 m, 1.0:4.3:4.7 at 140 m. and 1.0:5.0:6.8 at 950 m. DISCUSSION

WHISP sample from VERTEX III. Alkanes and sterenes were roughly equivalent in the sinking particles. Very pronounced suspended sterene concentration maxima were observed at the top of the oxygen minimum (200 m and 140 m for VERTEX II and III, respectively; Table 2). This contrasts markedly with all other steroid classes which had concentration maxima in the euphotic zone at the depth of the biomass maximum. There is a suggestion of a flux maximum for sinking sterenes in the O*-minimum (e.g. 100 m for VERTEX II), but insufficient samples were collected for verification. Nevertheless, sterenes were enriched relative to POC and sterols in both sinking and suspended particles in the OZ-minimum. The distributions of sterenes were complex (Figs. 8 and 9 and Table 6) with up to 24 compounds present representing CZ7, CZs, and &, monoenes, dienes, and trienes. The lack of authentic standards precluded detailed identifications in most cases. Cholesta-3Sdiene @J was most often the dominant component. Sterenes containing Azqzn unsaturation on the alkyl side chain were detected, while sterenes with AZ2 unsaturation were relatively few. VERTEX II PIT samples below the euphotic zone contained major amounts of a cholesta-N,N,24-triene (2) which was unaccountably absent from the VERTEX III PIT samples. Cholest-Z ene (5J was abundant in the euphotic zone WHISP samples from both years but its relative abundance decreased with increasing depth while cholesta-N,3.5triene (10) increased. Sterenes in the sediment floe consisted primarily of cholesta-3,5diene, cholestaN,N,ZCtriene (6J and 24-methylcholesta-N,24(28)diene (14). Mass spectra of the cholestatrienes (3, 9, and 2lJ suggest they are anthrasteroids (HUSSLERand ALBRECHT, 1983). In no samples were CJO-sterenes, A’- or AS-sterenes, diasterenes, A-ring monoaromatic steroids, or steranes found. In all samples, Cz7 sterenes dominated (Fig. 5); Cz7 sterenes were particularly abundant in most oxygenminimum and deeper PIT samples. In general, C2,

Steroid distributions presented above demonstrate that the two major particle size classes-sinking large particles and suspended small particles-are different in terms of organic matter source, transformation and transport. Most of the particulate matter is produced by organisms inhabiting the euphotic zone, so the organic composition reflects these predominantly biogenie origins. At the same time, 90% or more of the particulate organic matter is degraded within the euphotic zone, primarily through consumption and decomposition by the heterotrophs, thus modifying its organic composition (LEE and WAKEHAM,1988). Only about 5-109 of the particulate organic matter produced in the upper ocean is transported out of the euphotic zone, and the amount of organic matter in large and small particles decreases with depth due to continued decomposition and alteration. If there are differential source processes forming large vs. small particles and if the particle classes are subjected to different decomposition/alteration processes, then their compositions in mid-depth regions may be significantly different. Biological sources of steroids Sterol compositions of sinking and suspended particles reflect in part the different sources for organic matter in each size class. Large amounts of cholest-5en-36-01 and cholesta-5,22Edien-38-01 (3J are likely derived from zooplankton carcasses and feces since these two Cr, sterols dominate the sterols of many genera of marine animals (MORRIS et al., 1982, 1984, and reviews by MORRIS and CULKIN, 1977, and GOAD, 1978) and their fecal matter (PR4HL etal., 1984a, 1985; NEAL et al., 1986; WAKEHAM and CANUEL, 1986; HARVEYef al., 1987). Carcasses and fecal pellets are large and rapidly sinking particles which will be best collected in sediment traps but inefficiently sampled by the WHISPs. 24-Methylcholesta-5,22Edien-38_ol, 24methylcholesta-5,24(28)dien-3&ol, and 23,24dimethylcholesta-5,22Edien-38-01 are common in various species of planktonic algae (reviewed by

3062

S. G. Wakeham

VERTEX

II WHISPs DA2 BZlA315

PITS

q AN.22 goAN.N p AN.%@8) AN.3.5 pi AN.N.24(28) r-~ANaN,

20

ii2 TRIENES

0

2

4

6

8

10 12 14 16 18 20 22 24

2

4

6

BfO%2f416l8202224

8 (0 12 14 f6 l8 20 22 24 BEi

FIG 8. Distributions of sterenes in VERTEX II PIT. WHISP, and sediment (3500 mf samples. De&nations refer to Table 6. VOLKMAN,1986). The small amounts of 4,23,24-n% methylcholest-22E-en-3/?-oi and other 4-methylsterols indicate, however, that dinoflagellates, in which these sterols occur (ALAM et al.. 1979, 198 1; WITHERSfr al., 1978; BOON et al.,1979; VOLKMANef al.. 1984; ROBINSONer al., 1984; WENGROVITZet ai., 198 I) probably were not major contributors to particulate sterols. Algal cells will be incorporated into fecal pellets and marine snow-type aggregates which may be sampled by traps, or they may be free or in readily disag-

gregated marine snow which can be sampled by in s&r filtration, This interpretation is consistent with results for sterols (GAGOSIANef al.,1983; WAKEHAMet al., 1984a) and carotenoids (REPETA and GACXXIAN,1984) in the euphotic tone of the Peru upwelling area, and other lipids (WAKEHAMand CANUEL, 1987) at this VERTEX site, in which suspended particles contained more of a phytoplankton signature and less of a zooplankton signature than large, sinking particles. The stereochemistry at C-24 of 24-ethyicholest-5-

3063

North Pacific steroid geochemistry

VERTEX

III WHlSPs R A2 H A3q5 0

q AN.22 2

4

6

8

10 12 14 16 18 20 22 24

AN,N AN.24 (28)

PITS

(0

q AN.395

321 0

2

4

6

6

40 12 14 16 (6 2022

q AN,N.24 (28)

24

230

20

q AN.N.24

t--

50

2

4

6

8

IO 12 14 (6 18 2022

R TRIENES

24

0

10 (2 14 16 (8 2022

24

0

2

4

6

6

10 (2 14 16 18 2022

24

2

4

6

6

IO 12 14 t6 16 2022

24

“1

0

2

4

6

8

x) 12 14 16 (8 2022

24

0

I

95Om

2

4

6

6

(0 (2 14 16 (6 20 22 24

2

4

6

8

10 (2 14 16 18 XI22

24

FIG. 9. Distributions of sterenes in VERTEX III PITand WHISP samples.

en-3&ol has not been established, but it is likely that that 24-ethylcholesterol cannot be attributed solely to at least some of this compound is terrestrially-derived terrigenous sources since it may be abundant in areas &sitosterol. eqxcially in suspended particles. 24-Ethylremote from higher plant sources (MATSUMOTO etal., cholesterol has been recognized in some algal species 1982; VOLKMAN, 1986). However, the assignment of (PAOLE-ITI eral.,1976;VOLKMAN etal., 1981)and some portion of the 24-ethylcholesterol as &sitosterol clionosterol, the coeluting C-24 epimer of &sitosterol, in the VERTEX samples is consistent with the hydrois found in sponges and is considered a marine sterol graphic regime. MARTIN and KNAUER (1984) have (!3~~~~~~,1973).ThusV0~~~~~(1986)hi~stressed previously concluded that lateral advection off the

S.G. Wakeham

3064

Table 6. Sterenes essigned in VERTEX particle seraples.

Resignation (Figs. 6 and 91

Assfgnaent cholesta-N*,22-dlene cholesta-N.t2-diene C27-cholestetriene cholesta-N,24(28f-diene cholest-t-ene choiesta-N,N,tQ-trlene choleste-N.22-diene cholesta-3,5-diene C2R-cholestetriene cholesta-N,3,5-triene c~les~-N,N,24-triene 24"methylcholesta-N-24(28)-diene Zr)-eethylcholest-2-ene 24-methylcholesta-N.24(28)-diene 24-methylcholesta-N.N.24(28)-triene 24-olethylcholesta-3.5-dfene 24-~t~lc~lesta-N,N,24(28)-triene 24-ethylcholestene 24-wthylcholesta-N,N-diene 24-ethylcholest-2-ene C g-cholestatriene 2ri -ethylcholesta-N,24f281-diene 24-ethylcho~esta-3,5-diene 24-ethylcholesta-N~N,24(28~-triene

dist~butions in the VERTEX water column samples strongly implies an upper ocean input. SMITH eta/., (1982). however, caution that 3-methoxy steroids might be analytical artifacts. Extraction of WHISP samples with toluene-methanol might result in production of 3-methoxy steroids, but the methylene chloride extraction ofthe sediment trap samples should not. Thus while 3-methoxy steroids as artifacts in the WHISP samples cannot be ruled out, the presence of these compounds in the PIT samples suggests their existence in nature. Reports of sterenes in marine organisms are limited (HAMILTON~~ al.. 1975; WAKEHAM and CANUEL. 1986). and like the steroidal ketones their origin is unclear. In situ alurations of particle composition

In situ alteration processes can change the organic composition of particles in three ways. 1) There may be preferential removal of some dietary components leaving other components dominating. 2) New compounds may be produced by conversion of dietary material or biosynthesized de nova utilizing elements and energy obtained by feeding. 3) Biochemical transformations may yield completely new compounds. Su*N = nucl ar unseturatfon,positfon unknown but not ~~ ~~m~sition of these processes on the commotion or d s. of material exiting the euphotic zone should result in organic compound distributions observed at depth. Mexican continental shelf leads to 7& of the dissolvedZooplankton and fish alter the organic composition of particles they ingest. Alterations occur via enzymatic manganese maximum in the oxygen minimum zone reactions during assimilation or by transformations by at this location. Thus it is possible that terms&l sterols enteric microflora. In both cases, the end product will are likewise transported laterally as suspended particles, be fecal matter having a composition which may be resulting in some of the elevated abundances of 24 ethylchol~terol in and below the oxygen minimum. significantly different from that of the animal’s diet. Biological sources for other steroid classes present Most organic matter in fecal material represents residin the VERTEX samples are less well defined, mainly ual dietary material which has not been assimilated, because there have been substantially fewer reports of as well as compounds endogenous to the animal. Hertheir distributions in marine organisms than for sterols. bivores can produce fecal pellets which contain extenIn addition, the quantitative inputs to the particulate sively modified algal lipids in addition to compounds organic matter pool by these other organisms is gennot present in the algal diet but biosynthesized by the erally unknown. Low levels of stanols have been idenanimal (VOLKMAN~~&., ~~~~:PRAHL etal., 1984a,b; tifiedinsomealgae (CF~ARE~~N-LORRIAUX~~U!., 1976; HARVEY et al.,1987). Porexample,phytosterols such NIS~IMU~ and KOYAMA, 1977), but significant as 24-methylcholes~-S,22Edien-3~-ol and 24_methamounts of stanols have been reported in marine inylcholesta-5,24(28)dien-3j3-ol are preferentially devertebrates, including molluscs, annelids, sponges. tupleted while cholesterol is enriched in fecal pellets nicates, and coelenterates (MORRISand CULIUN,1977: compared to the algae. Dietary fatty acids appear to GOAD, 1978). In addition to dino~agellate sources. 4- be more readily metabolized than dietary sterols metbylsterols may also be produced by some bacteria (PRAHL cl al., 1984a; WAKEHAM and CANUEL, 1986; (BIRD et al., 1971; BOUVIER et al., 1976). Steroidal NEAL et al..1986; HARVEY ef al.,1987), resultingin ketones are generally not thought to be significant confecal pellets enriched in sterols and depleted in fatty stituents of marine organisms, although they are inacids. The feeding m~hanism can also influence which termediates in sterol biosynthesis (GOAD, 1978). A”- compounds are preserved or destroyed (NEAL etal., Stenones have been reported in red alga (KANAZAWA 1986); fecal pellets produced during herbivorous feedand YOSHIOKA, 197 1) and dinoflagellates (KOKKE et ing were compositionally different from pellets proal., 1982; WITHERS efal., 1978). Stenones and stanones duced by coprophagy. have been found in several marine animals ( POPOV et Zooplankton can convert dietary material to meet al., 1976; EDMONDS~U!., ~~~~;DEESETH etal.,1978; specific metabolic needs. For example, it is well estabWAKEHAM and CANUEL, 1986kbutitis not known lished that zooplankton dealkylate dietary Czs and Cr9 whether they are natural products or microbial aherphytosterols to produce C,, cholesterol, which it is ation products of dietary sterols. No biological source thought that they are unable to biosynthesize de novo. for 3-methoxy steroids has been reported, but their Copepods will often biosynthesize large amounts of

3065

North Pacific steroid geochemistry energy reserve wax ester5 from assimilated dietary fatty

acids. Thus, animals can generate compound distributions which are very different from the material they ingest, and mesopelagic organisms may be able to produce lipid compositions considerably different from their epipelagic cousins. Such processes have been implicated in the mesopelagic flux maxima which have been reported for sterols (WAKEHAMet al., 1980; GAGOSIANet al., 1982) and wax esters (WAKEHAMet al., 1984~) in the open ocean. Mesopelagic production of lipids can also account for the observation that deep water particles appear fresh and cannot merely rep resent degraded material from the surface (WAKEHAM et al., 1984~; WAKEHAMand CANUEL, 1987). Microbial transformations of organic material may also produce new compounds. Transformations of dietary material by gut microorganisms may introduce transformation products into feces. For example, PRAHL et al. (1984b) detected dihydrophytol in fecal pellets of Calanus fed on a alga1 diet deficient in dihydrophytol. Biological reduction of algal phytol ap peared to proceed both microbially by gut microflora and enzymatically by the animal itself. Steroidal hydrocarbons and ketones present in fecal pellets of a pelagic crab have also been attributed to microbial transformations in the gut of the crab (WAKEHAMand CANUEL,1986). Microbial alteration of biogenic stenols to stanols, steroid ketones, and sterenes occurs in Recent sediments, most likely by a series of oxidation, dehydration, and reduction reactions (reviewed by GAGOSIANm al., 1980 and MACKENZIEet al., 1982). A’-Stenols are progressively oxidized and hydrogenated via ketone intermediates to stanols and monosterenes; steradienes may form directly from the A5-steno1 precursors and steratrienes from A’-stenols via steroid diol intermediates (Fig. 10). Recent evidence (MERMOUD et al., 1984) has also shown a direct steno1 * stanol conversion in anoxic lake sediments. Anaerobic bacteria are capable of converting stenols to stanols (BJORKHEMand GUSTAFSSON, 197 1; EYSSEN et al., 1973; PARMENTIERand EYSSEN, 1974; GASKELLand EGLINTON,1974; TAYLORet al., 1981) with A4-3-ketostenones and 3-keto-stanones as intermediates. A4Stenones may be formed in large particles as they sink through the oceanic water column (GAGOSIANet al., 1982). However, conversion of A’-stenols to Sa-stanols was not observed either in the open ocean (GAGOSIAN et al., 1982) or at the oxicfanoxic interface ofthe Black Sea (GAGOSIANand HEINZER, 1979). Production of sterenes in suspended particles at this VERTEX site was previously suggested by WAKEHAMet al. ( 1984b). Steno1 compositions in sinking and suspended particulate matter at VERTEX II/III are most likely controlled by a combination of surface water phytoplankton and zooplankton production, mid-water production of zooplankton sterols by animals inhabiting the mid-depth regions and consuming living and detrital particles, and production of fecal matter containing extensively modified dietary sterols. Since the major steroid classes involved in the proposed steno1 -+ ste-

roidal ketone --c stanol+ sterene conversion pathways (Fig. 10) have been determined in sinking and suspended particles at VERTEX II and III, is there evidence for in situ transformations in the water column? If so, do the two particle classes show similar or different behaviors? To address this question, vertical distributions of ratios of potential transformation products to the major precursor, cholesterol, in VERTEX PIT and WHISP samples are shown in Fig. 11. Values for the sediment floe are given in the VERTEX III panels. Similar trends were observed for other steno1 precursor/ product pairs. There is a marked enrichment of cholest4-en-3-one, Sa- and 5&cholestan-3-one, Sa-cholestan01 and (cholest-2ene + cholesta-3,5diene) in suspended particles in the oxygen minimum zone, and in the case of cholest&m-3-one, below the oxygen minimum zone as well. Product:precursor ratios in the sinking particles are always substantially lower, and maxima in the Oz-minimum zone less apparent. Four explanations for the enhanced abundances of sterones, stanols, and sterenes are possible, but three are unlikely. First, specific inputs of these steroid ciasses from organisms inhabiting the oxygen minimum zone seems unreasonable. The oxygen minimum is characterized by a conspicuous lack of living zooplankton (M. TUEL and M. SILVER,pets. commun.) which might be likely steroid sources. Second, preferential in situ

,o& “;* sa(HWmm8

9”

SaDi) Smnoi

i A’*’ .SmraOmm

5&H) Stonol

% A*

Slrme

smmbm

FIG. 10. Steroid transformation pathways, adapted from GAGOWN and FARRINGIDN (1978), GAGOWNet al. ( 1980),

and MACKENWE ef al. (1982).

3066

S. G. Wakeham

VERTEX

VERTEX

If ;~;:;~:;jt

IU

VERTEX

II

VERTEX IIL

5qi?-cwM3~ Cholest-~efl-3B-o/

c%J

1

,SEDfhlENT

0

18

0

26

RG. I 1. Pnxiuct:precursor ratios (as %) for VERTEX II/III PIT, WHISP, and sediment (3500 m) floe

samples.

microbial or chemical degradation of the precursor steno1 relative to product would increase in product: precursor ratio. The site of such microbial degradation would be the oxygen minimum zone where there is enhanced microbial activity. Furthermore, if the suspended particles have longer residence times in the oxygen minimum than sinking particles, then ratios in suspended particles could easily be greater than for sinking particles, as in fact is observed. However, previous results suggest the lack of differential in situ water column degradation of either 114-stenones (CAGOSIAN et al., 1982) or stanols ~GA~IAN and HEINZER, 1979) compared to steno]. On the other hand, preferential degradation of stenols relative to stanols has been indicated in sediments (NISHIMUIU AND KOYAMA, 1977).

Third, it could be argued that the increased abundances of the diagenetic products in the oxygen minimum zone simply result from lateral advection of pimento material, especially in the suspended particle size range, which aheady contains these compounds, Several aspects of the steroid distributions are inconsistent with this hypothesis as well. The manganese data of MARTINand KNAUER( 1984) indicate that the lateral transport of continental shelf-derived

material delivers manganese to this location in the 200400 m depth interval. It should therefore be in this depth range that steroids having sedimentary origins should be consistently most abundant (i.e., product: precursor ratios the highest). However, there is no consistent pattern to the ratios. On the contrary, three patterns are apparent: stanone:stenol and sterene:stenol ratios are highest at 140-200 m; stanoI:stenol ratios maximize between 140 and 500 m: and stenone:stenol ratios increase from the surface to 1500 m. Furthermore, product:precursot ratios in suspended particles in the oxygen minimum tended to be consistently higher than the sediment values we measured and particle compound distributions were different from the sediment floe. However, there is of course no murance that the sediment bloc under the VERTEX Ii/III site has the same steroid composition as sediment on the continental shelf The remaining and most likely hypothesis is that there is active transfo~ation of stenofs yielding the complete series of steroidal ketones, stanols, and sterenes in particles in the oxygen minimum zone. Moreover, this transformation seems to occur primarily in suspended particles. The long residence time of suspended particles in the Or-minimum compared to

3061

North Pacific steroid geochemistry rapidly sinking particles may play a major role in increasing the relative abundances of the products over their abundances in sinking particles. Microbially-mediated processes are probably involved in these water column reaction sequences, as they are in sediments. Intense microbial decomposition of suspended and sinking particulate organic matter in the 100-600 m depth interval at VERTEX II/III has been suggested by LEE and CRONIN (1984) to result in an inverse relationship between dissolved oxygen and molar concentrations of amino acid omithine, a microbial decomposition product of arginine. KARL and KNAUER (1984) conducted in situ incubation experiments during VERTEX II and III to assess microbial activity and growth in the water column and found intensified microbiological processes in the oxygen minimum, especially between 700 and 900 m. This depth range is, however, at the base of the oxygen minimum and is below the interval in which most steroid transformation apparently occurs. KARL,and KNAUER also found that sinking particles contained a much more active microbial community than suspended particles, and GOWING and SILVER( 1983) found greater abundances of bacteria in fecal pellets from sediment traps than in suspended particles. The steroid results, on the other hand, indicate enhanced transformations on suspended particles. CONCLUSIONS 1. Sterols, 3-keto-steroids, 3-methoxy-steroids and sterenes are present in sinking and suspended particles in the eastern tropical North Pacific. The major source of steroids associated with particulate matter is biological production in surface waters. In situ decomposition of particulate organic matter leads to the ob served decreases in the vertical flux of sinking particulate steroids and suspended steroids. 2. Sinking particles tend to be enriched in zooplankton-derived sterols but depleted in phytosterols and termsuial sterols compared to suspended particles. The compositional differences reflect differences in both the sources for sinking and suspended particles and the degree of decomposition which alters the organic composition of the two classes. Sinking particles appear to represent material which has been more extensively modified, probably by herbivores and coprophagic feeders, while suspended particles seem to be less degraded by macroheterotrophs. 3. Relative abundances of steroid ketones, stanols, and sterenes vary considerably as a function of particle size and sample depth. Steroidal ketones, stanols, and sterenes are all substantially enriched in suspended particles in the oxygen minimum zone. Suspended particles in the intense oxygen minimum zone appear to be the site of active steno1 + steroidal ketone + stanol + sterene conversions; these conversions are most likely mediated by the active microbial community inhabiting this region. Steroid transformations appear less significant in sinking particles, but this could

result from their shorter residence times in the oxygen minimum. AcknowZedgemenfs-C. H. Clifford, K. Bruland, K. Coale, J. Cowen, and G. Smith helped in collectingthe PIT and WHISP samples. J. Livramento and E. Canuel aided in laboratory analyses, and N. Frew assisted in gas chromatography-mass spectrometry. I thank J. W. Fanington and R. B. Gagosian for thoughtful discussions. Support for this research came through the Oflice of Naval Research via contracts NO001485C-0071 and Nob1487K-0071 and National Science Foundation grant OCE 80-03200 for VERTEX. Editorial handling: J. W. de Leeuw REFERENCES ALAMM., SANSINGT. B., BUSBYE. L., MARTINEZ D. R. and RAYS. M. ( 1979) Dinoflagellatesterols 1:stem1composition of the dinollagellates of Gonyaulas species. Steroids 33, 197-201. ALAM M., SANSINGT. B., GUERRAJ. R. and HARMON A. D. ( 1981) Dinoflagellate sterols IV: Isolation and structure of 4cr,23E,24E-trimethylcholestanol from the dinoflagellate Glenodinium hallii. Steroids 38, 375-382. BIRDC. W., LYNCHJ. M., PIRTS. J., REIDW. W., BROOKS C. J. W. and MIDDLEDITCH B. S. (197 1) Steroids and soualene in Methylococcuscapsukatw&own &I methane. No&e 23q473-474. BJORKHEM I. and GUSTAFSSON J.-A. (197 1) Mechanism of microbial transformation of cholesterol into coprostanol. Eur. J. Biochem. 21,428-432. Boon J. J., RIJPSTRA W. 1. C., DE LANGF. and DE LEEUW J. W. (1979) Black Sea sterol-a molecular fossil for dinollaaellate blooms. Nature 277. 125- 127. BOUVIE~P., ROHMERM., BENEV& P. and OURI~~ONG. (1976) Aso’)-Steroidsin the bacterium Methylococcuscap sulafus. Biochem. J. 159,267-271. BRASSELL S. C. and EGLINTON G. (1983). Steroids and triterpenoids in deep sea sediments as environmental and diagenetic indicators. In Advances in OrganicGeochemistry 1981 (eds. M. &OROY etal.), pp. 684-697. John Wiley. BROENKOW W. and KRENZR. (1982) Oceanographic results from the VERTEX II particle interceptor trap experiment off Manxanillo, Mexico, 26 October to 18 November 1981. In Moss Landing Marine L&oratories Tech. Pub. 82-l. BROENKOW W. W., LEWITUS A. J. and REAVESR. E. (1983a) Oceanographic results from the VERTEX 3 particle interceptor trap experiments off central Mexico, Gctober-December, 1982. In Moss Landing Marine L&oratories Tech. Pub. 83-l. BROENKOW W. W., LEW~TUS A. J., YARBROUGH M. A. and KRENZ R. T. (198313)Particle fluorescence and bioluminescence distributions’inthe easte.mtropical Pacific. Nature 302,329-33 1. CHARDON-LORRIAUX I., MORISAIUM. and IKEKAWAN. (1976) Stem1 profiles of red algae. Phytochem. 15, 723725. DEISETHC., CARLSONR. M. K., DJERASSIC., ERDMANN T. R. and SCHEUER P. J. (1978) Identification de sterols a chaines laterales courtes dans l’eponge Damiriana hawaiiana.He/v. Chim. Acfa 61, 1470-1476. EDMONDSC. G., SMITHA. G. and BROOKSC. J. W. (1977) Analysis of sponge sterols as the trimethylsilyl ethers and as the corresponding 5a- and A’-3-ketosteroids using open tubular gas chromatography-mass spectrometry. Application of selective enzymic oxidation. J. Chromatogr. 133, 372-377. EY~~ENH.J.,PARMENTIERG.G., COMPERNOLLE F.C.,DE P~~wG.andPlEssENs DENEFM. ( 1973) Biohydmgenation of sterols by Eubacterium ATCC 21,408-Nova species. Eur. J. B&hem. 36,421-431.

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