Lipids of aquatic organisms as potential contributors to lacustrine sediments—II

Org. Geochem. Vol. 11. No. 6, pp. 513-527. 1987 Printed in Great Britain. All rights reserved

0146-6380/87 $3.00+0.00 Copyright © 1987 PergamonJournals Ltd

Lipids of aquatic organisms as potential contributors to lacustrine sediments--ll* P. A. CRANWELLI, G. EGLINTON 2 and N. ROBINSON" tFreshwater Biological Association, The Ferry House, Ambleside, Cumbria LA22 0LP, U.K. ?Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 ITS, U.K. (Receired 10 November 1986; accepted 21 April 1987)

Abstract--The relationship between the lipid composition of organisms in the water column of an eutrophic lake and the lipid composition of underlying sediments, previously examined for n-alkanols and steroids, is now reported for hydrocarbons, ketones and carboxylic acids. The n-C,~ alkane and alkenoic acids from two primary sources are rapidly metabolized in the water column and surficial sediment. Bacterial biomarkers, including hopenes and i/ai-branched fatty acids, were detected in the photosynthetic bacterial layer occurring just above the sediment-water interface. Within the sediment the apparent conversion of free n-alkanes, alkan-2-ones and co-hydroxy acids to the corresponding bound form is noted; microbiological oxidation of n-alkanes to alkan-2-ones is supported by the detection of the intermediate alkan-2-ols with a distribution similar to that of the ketones. The geochemistry of sediment deposited c. 1900, prior to biological study of the site, was interpreted from stable biomarkers and the diagenetic changes recognised in the study of contemporary deposition. A qualitative difference in algal input to the older sediment is inferred from the low ALsterol content and presence of 2,6,10-trimethyl-7-(3-methylbutyl)-dodecane. However, there was still significant dinoflageilate input, as indicated by the presence of 4x-methylsterols. A difference in higher-plant input to the older sediment, indicated from the n-alkane, alkene and triterpenoid ketone distributions, is consistent with the recent development of tree cover. Key words: biological markers, lipids, hydrocarbons, carboxylic acids

lipids in the water column was studied by analysis of populations of rotifers, ciliated protozoa and photosynthetic bacteria that are stratified into zones, each of low species diversity and high biological density, fulfilling the ecological requirements of the respective organisms (Robinson et al., 1984b). Determination of the lipid composition of these organisms and of the surficial sediments enables the processes occurring between synthesis by the primary producers and incorporation of residues into the sediment to be studied, as shown by analyses of steroidal compounds (sterols, stanones and sterenes) and nalkanols, previously reported (Robinson et al., 1984b). We report here non-steroidal hydrocarbons and ketones and also monocarboxylic acids and hydroxyacids of these organisms and surficial sediment. Short-chain lipids characteristic of aquatic primary producers undergo further breakdown in the uppermost sediments (Cranwell, 1984a), thus chain length distributions of homologous series of free lipids in a sub-surface sediment (deposited c. 1965--1974) were studied to detect such processes, also free sterols were determined, to recognise input from aquatic biota. In relation to the assessment of input to older sediments, the value of these methods of studying the fate of lipids during deposition is demonstrated by analysis of the same lipid classes (steroids, alcohols, hydrocarbons, ketones and fatty acids) of a deeper sediment section deposited c. 1900-1910, for which there

INTRODUCTION Knowledge of the sources and fates of biolipids in a variety of modem depositional environments, including marine, freshwater and hypersaline systems, is relevant to assessment of the depositional history of ancient sediments based on their geolipids. Methods for studying the initial changes in lipid composition of primary biota during sedimentation through the marine water column include (a) laboratory studies of the food web, in which the lipid content of zooplankton faecal pellets is compared with that of a specific phytoplankton food source (Prahl et al., 1984), (b) cultivation of marine bacteria using a specific natural product (e.g. phytol) as sole carbon source (Giilan et aL, 1983a) or (c) deployment of sediment traps throughout the oceanic water column (Wakeham et al., 1980). Direct sediment input from a primary aquatic source organism may be recognized by a correlation between the distribution of a suite of stable biomarkers in the source and sediment, as exemplified by the use of 4cc-methylsteroids to recognize dinoflagellate input to the sediment of Priest Pot, a small (area, 104 m-'; max. depth, 3.94 m), highly productive (summer chlorophyll a levels up to 2.3 m g - ! - ' ) lake (Robinson et al., 1984a). In this lake the fate of

*Paper I: Org. Geochem. 6, 143-152 (1984). O G . I 1:6--F

513

P.A. CRANWELLet al.

514

is no direct evidence about contemporary aquatic biota. EXPERIMENTAL

Sample collection Sediment samples were obtained with a pneumatic corer l m in length (Mackereth, 1969). Section 1 (Table l) and incompletely analysed section la (8-14 cm) deposited c. 1965-1974 were removed from a core taken in August 1981, while section 2 came from a core obtained in 1983, dated by measurement of unsupported 2~°Pb, assuming a constant rate of supply (Oldfield and Appleby, 1984). Natural populations of rotifers, ciliated protozoa and photosynthetic bacteria were collected in July 1982, as described previously (Robinson et aL, 1984b); the main species of photosynthetic bacterium, previously assigned to the genus Chlorobium, has now been identified as Clathrochloris hypolimnica (Chlorobiaceae) (Davison and Finlay, 1986).

Extraction and analysis Extractable and bound lipids were isolated from sediments as previously reported (Cranwell, 1978). Isolation and fractionation of lipids from the organisms was previously reported in paper I (Robinson et al., 1984b). The alcohol, sterol and hydroxyacid methyl ester fractions were derivatized with BSTFA prior to gas chromatographic analysis under the conditions previously reported. C-GC-MS analysis and methods of identification and quantitation of individual components have been described in Part I.

abundances of both free and bound lipid components are given, also abundances of the compound classes isolated from the organisms. For each compound class, results are presented for organisms prior to those for sediments.

Hydrocarbons The rotifer lipids contained relatively low levels of n-alkanes of a unimodat, smooth distribution maximising at C:4 (Fig. 1). n-Alkanes of both ciliates and C. hypolimnica were dominated by n - C i 7 , with the C, hypolimnica alkane distribution also containing a much smaller maximum a t n - C 2 9 and relatively low amounts of hopanoid hydrocarbons, which had a distribution similar to those isolated from the uppermost sediment (see Table 2). Alkenes were present in the ciliates and photosynthetic bacteria. In the former, isobotryococcene (1), identified from its mass spectrum, relative retention time and those of its hydrogenation product, was present at an abundance c. 170% that of n-CL~ alkane. Squalene was present amongst the lipids of C. hypolimnica. The free and bound sedimentary n-alkane distribution are also shown in Fig. I, together with the abundance of the major branched-chain alkanes,

1

RESULTS 2

The elemental composition of sediment sections 1, la and 2 is given in Table I; for sections l and 2,

Scheme 1. Structural formulae.

Table I. Composition of organisms and sediments Organisms Sample

Depth ~ (cm)

Rotifers

50

Ciliated protozoa

150

Bacteria

380

Abundances~ Lipid Hydrofraction carbons

Main species present

Keratellacochlearis, K. quadrata Polyarthra spp. mainly rulgaris ; Anuraeopsis fissa Loxodesmagnus;L. striatus Spirostomum spp.; Prorodon sp.; Frontonia sp. Mainly Clathrochloris hypolimnica;

Acids

Hydroxy Desmethyl 4~t-Methyl acids Alkanols sterols sterols Ketones

total

1.4

6.4

ND

6.2

4.4

0.8

ND

total

17.0

ll6.0

ND

10.0

5.1

0,2

ND

free

10.5

127,0

1.4

88.0

74.0

32.0

3,4

free bound

76,0 1.2

30.4 130,0

0.5 41.0

250.0 6.5

203.0 10>l

I I 1.0 1.3

24.7 ND

free bound

23.0 17.5

0.6 65.0

0.5 28.4

220.0 81.0

88.0 8.3

117.0 1.8

7.5 5.1

Some purple photosynthetic bacteria Sediments c Section

Depth a (cm)

Age (years)

C (%)

H (%)

N (%)

I

0--6

0-5

22,0

2.6

2.0

la 2

8-14 51-59

7-16 73-83

19.0 16.1

2.3 2.1

1.8 0.9

aMeasured in water column (organisms) or in sediment column. ~As #g/I water for organisms, or as ,ug/g dry. extracted sediment. N D - - n o t detected. Ketones = n-alkan-2-ones. Abundances reported in Part I for sediment section I were based on an incorrect weight of sediment; corrected values are given here, ~Sections I and la from core collected 1981: section 2 from 1983 core dated using a°Pb, assuming constant rate of supply model.

Lipids of aquatic organisms *eo] (A)

,

ngl-

as a partially reduced squalene, recently detected in sapropel from a playa lake (Albaiges et al., 1984).

='1 Rotifers ~tote,

,lllltlt,,

] 4700" ](B, ~ 7

"

Ketones 6,10,14-Trimethylpentadecan-2-one occurred in the rotifers and in C. hypolimnica (8 and 300 ng. i-', respectively); the latter also contained minor amounts of hopanoid ketones. Straight chain alkan-2-ones showing a high CPI were identified in the free lipids of sediment section I and in the free and bound lipids of section 2 (Fig. 2). 6,10,14-Trimethylpentadecan-2-one was abundant in the free lipids of the uppermost sediment (840 ng.g -t) and in the bound lipids of section 2 (720 ng. g - ' ) . Pentacyclic triterpenoid ketones characteristic of higher plants were also abundant in sedimentary free lipids (Table 3).

' Ci,iet,, ,toter,

°°1,I11

400

h,,,,,, ....

I "24ro "

ng 1-1 ](C) ~

"

Photosynthetic Bacteria

,..s.

°°1 '1' Se,',ment t

I "oo'llll oo" Ph

Monocarboxylic acids

10

2.5

,-1 I';

"°":;

l]

15

20

25

30

515

.ol -o'o.o,,ll

,.ol

uoo III

35 15 20 Carbon Number

25

30

Fig. 1. Abundances, expressed as ng-I water-' or pg.g dry sediment-', of n-alkanes in (A) Rotifer total lipids (B) Ciliated protozoa total lipids (C) Clathrochloris free lipids (D) Sediment 1 free (left) and bound (right) lipids (E) Sediment 2 free (left) and bound (right) lipids. Pr -- pristane Ph -- phytane X -- 2,6,10-trimethyl-7-(3-methyibutyl)dodecane. Abundances of free alk-l-enes in sediments are depicted as hatched bars to the left of the n-alkane having some carbon number; in (E) alkene abundances are magnified by a factor of 4, for clarity.

pristane and phytane in section 1 and 2,6,10-trimethyl-7-(3-methylbutyl)-dodecane in section 2. Hopanoid hydrocarbons were identified amongst the free lipids of sediment sections ! and 2 (Table 2). The free hydrocarbons of sediment section 2 also contained low levels (c. 100 ng.g -l) of fern-8ene and fern-9(1 l)-ene and six compounds tentatively identified as A-ring degraded triterpenoidal hydrocarbons. By mass spectral comparison with a standard (Hoffman, 1984), one of these constituents was identified as des-A-lupane (100 n g - g - ' ) . The free hydrocarbons of both sediment sections 1 and 2 contained a series of n-alkenes (C17-C27) maximising at C25 and showing odd-carbon predominance (Fig. l). The GC retention data (Sojak et al., 1980) and Rf value during argentation TLC (Brieskorn and Beck, 1970) suggest a terminal double bond. The surficial sediment also contained, among the free alkenes, squalene and three C30:6 alkenes, which, from their mass spectra, are probably isomers of squalene and a C3o:, alkene, tentatively identified

n-Alkanoic acids were detected in all samples; their distributions in organisms and in sediments are given (Fig. 3). Iso- and anteiso-branched fatty acids, m a i n l y i-Cl3_t7 and ai-Ct3js,, r w e r e trace constituents of the rotifers but showed higher relative and absolute abundance in the ciliates (9% of saturated acids, 4.2/tg-l-t), C. hypolimnica (16%, 9 . 4 p g - l - ' ) and sediment 1 bound acid fraction (19%, 17.8 p g - g - ' ) . Phytanic acid was a significant component of free acids in C. hypolimnica and surficial sediment (4.4/tg-l-' and 1.05/~g.g -t, respectively). Trace levels of 17#(H),21fl(H)-bisbomohopanoic acid were identified amongst the free and bound lipids of sediment 1, relatively higher amounts (300ng.g -~) were detected in the bound acids of section 2, together with the 17~(H),21#(H)- and 17# (H),21 = (H)-stereoisomers (70 and 40 ng. g- ', respectively). Table 2. Abundances of free hopanoid hydrocarbons in the sediments Abundanceb Compound"

22,29,30-Trisnorhop- ! 7(2 I)-ene C27 Hopene 17p(H),2 LB(H)-22,29,30-Trisnorhopane 30-Norhop- 17(21 )-ene Hop- 17(21)-ene ! 7•(H),21,, (H)-30-Norhopane 17=(H),21B(H)-Hopane C3o Hopene 17p(H),21 ~(H)-30-Norhopane ! 7,8(H),2 I=(H)-Hopane 22S- 17,,(H),21,8(H)-Homobopane 22R- 17~,(H),21 ~ (H)-Homohopane 17~(H),21p(H)-Hopane Hop-22(29)-ene Hop-21-ene Homohop-29(3 I)-enc 17p(H),21 p (H)-Homobopane 17p(H),21 p (H)-Bishomohopane

Section I

---90 60 -130 30 -20 I0 30 -2200 220 -130 Tr.

Section 2

160 50 140 270 130 130 80 -320 Tr. Tr. 500 70 1300 570 I O0 300 50

"Compounds identified by comparison o f mass spectra and relative

retention times with published data. Traces of C3~--C;5 hopanes were also detected by mass fragmentography. bExpres,~'d as ng.g dry extracted sediment -f and determined by comparison of GC peak areas with that of a known amount of n-C a alkane. - - Not detected; Tr. trace levels present.

P . A . CRANWELL et al.

516 29

Sediment

1200.

Free

Bound

Alkenoic acids were present in all samples, their abundances are given in Table 4.

1

(nOt detectedl

Iqydroxy acids 800"

Hydroxy acids were very minor constituents of the lipids of the organisms and were not investigated further. Although 2- and 3-hydroxy acid isomers co-eluted when analysed by GC as methyl ester, TMS ethers on OV-I liquid phase, relative proportions could be estimated from the mass spectra (Eglinton et al., 1968). The free lipids of the uppermost sediment contained < 150 ng.g -~ of Cs-C:s 3-hydroxy acids maximising at n-C~4 and having a high proportion of iso- and anteiso-branched species; trace amounts of Cj6-C:s 2-, ~o- and (co-l)-hydroxy acids were also present. The free lipids of sediment 2 contained even carbon number a~-hydroxy acids (Ct4-C:8), maximising at el6 and C,, (80ng'g -I each). Hydroxy acids were relatively abundant constituents of the bound sedimentary lipids. The distributions of bound 3- and a~-hydroxy acids are shown in Figs 4A and 4B, respectively. Bound C~s-C,6

400.

ng

g '

...

i

,

! , 27

600-

Free

,oo

J :

lb

23

I

"~ = 2b

!

-

2'5

3o Carbon

iiLii., ~5

20

.i,i1 Sound

i t ,

~

3o

I

Number

Fig, 2. Abundances of alkan-2-oncs in sediment t free lipids and in sediment 2 free (left) and bound (right) lipids. Co-occurring alkan-2-ols in sediment 2 free lipids are shown as broken lines. (a) = 6,10,14-trimethylpentadeean-2-one.

ng

16

3200, 24(]0. I -~ 1600,

IA) Rotifers (total)

18

80020-

I,.I {B)

is. 10.

I

16

Ciliates (totall

pg I -~

II ! Bacleria (free)

15

1

,o

6 ,.,JilH

,

,...

.,

16 {DI Sediment 1

9'

45

16

8-

Pgg-~ 5 4. 3 2

I I,!, ,;,i[ 160. 14(] ng

I.,

J_,

IE) Sediment 2

,

I 6

, 16

I,I. Bound

g '9~iI6(]3

ts

I ,2 o i J,II 2s

3o Carbon

IJ ~

..,20

2"s

I .i 3"o

Number

Fig. 3. Abundances of alkanoic acids in: (A) Rotifer total lipids (B) Ciliated protozoa total lipids (C) Clathrochloris hypolimnica free lipids (D) Sediment I free (left) and bound (right) lipids (E) Sediment 2 free (left) and bound (right) lipids. Thick bars represent n-acids, thin bars =iso-branched, broken bars = anteiso-branched, dotted bar = phytanic acid.

Lipids of aquatic organisms Table 3. Abundances of free triterpenoid ketones detected in organisms and sediments

Abundance Sedimenth Compound

C.h.°

I

2

10 ND Tr. ND ND

240 ND 340 20 30

30 60 250 ND 20

140 I00 470 70 80 800

410 ND 140 150 860 I 100

Hopanoidr 22,29.30-Trisnorhopan-21 -one 30-Norhopan-22-one Hopanone Bishomohopanone

Tfishomohopanone

Higher plant triterpenoids Taraxer-14-en-3-one Lup-22(29)-ene-3-one' Olean- 12-en-3-one Urs- 12-en-3-one Lupan-3-one Friedelan-3-one

ND ND 150 100 ND ND

=C.h. = Clathroehloris hypolimnica; Quantitation expressed as ng-I water- ~, determined by comparison of GC peak areas with that of a known amount of n-C2s alkane. hQuantitation expressed as ng.g dry, extracted sediment-~, determired as above. ND = Not detected, Tr. = trace levels. "Tentative assignment based solely OR mass spectral interpretation.

Table 4. Abundances of alkenoic acid in organisms and sediments Organismsh Alkenoic acid= Rot.

Sediment I ~

Cil.

Clath.

14:3 14:1 15:1 16:4 16:2 16:3 16:1 16:1 16:1

-------160 --

80 40 -5400 II00 1200 300 6000 140

. 890 1700 . . . -23,800 --

18:2

---160 -------

-10,800 10,000 6000 4200 460 1900 1000 200

18:2 18:3 18:1 18:1 20:4 20:4 20:3 20:1 20:1

8100

.

Free . . . .

.

3900 -. . . -.

. . . .

. . .

.

1100 3200 -. . . 90 .

Bound

. . .

. .

. . . . . . . . . 1200 12,800 80 120 --130 1900 --

"~ j

Bound Free

. . .

Sediment T

.

517

c o - e l u t e d w i t h t h e 1,14- a n d 1,13-diol i s o m e r s a n d w i t h t h e C30 a l k a n - 1 5 - o n e - l - o l . T h e a b u n d a n c e a n d d i s t r i b u t i o n o f free s t e r o l s in s e c t i o n l a a n d b o t h free a n d b o u n d s t e r o l s in s e c t i o n 2 are compared with the corresponding components o f t h e surficial s e d i m e n t in Fig. 6; d a t a f o r s t e r o i d a l h y d r o c a r b o n s a n d k e t o n e s in s e d i m e n t s I a n d 2 a r e c o m p a r e d in T a b l e 6. W a x e s t e r s m a x i m i s i n g in a b u n d a n c e a t C ~ occ u r r e d in all s e d i m e n t s ( T a b l e 7). T h e m o l e c u l a r compositions of corresponding homologues from sediments i and la agreed closely but these generally differed f r o m t h o s e in s e d i m e n t 2. DISCUSSION Initial r e s u l t s c o m p a r i n g t h e lipid c o m p o s i t i o n o f a q u a t i c o r g a n i s m s a n d t h a t o f surficial s e d i m e n t using hydroxylic and steroidal constituents gave useful i n s i g h t i n t o t h e s o u r c e s a n d fate o f lipids in p r e s e n t d a y s e d i m e n t a r y s y s t e m s ( R o b i n s o n et al., 1984b). D i s c r e t e i n p u t s o u r c e s (e.g. o f h e x a d e c a n - 2 - o l a n d f a r n e s o l ) , d i f f e r e n c e s in s t a b i l i t y (e.g. loss o f C~2-C18 n - a l k a n o i s , A 5"7- a n d m o r e h i g h l y u n s a t u r a t e d s t e r o l s , b u t e n r i c h m e n t o f AT-sterols w i t h i n c r e a s i n g d e p t h in t h e w a t e r c o l u m n ) a n d m i c r o b i a l l y m e d i a t e d Sediment t

14

B

16

----

16

..[.,I1,,I..

.

3800

--

--

4700

--

--

8800 5200 . . . -.

---

400 100

. . .

22

ii

S

3

1[ Sediment 26

~ug g'~

It

16

2

--

22

--

,111,

.

"Position of double bonds not determined. SRot = Rotifers; Cil. = Ciliated protozoa; Clath. = Photo-synthetic bacteria. Quantitation expressed as ng.I water-J, determined by comparison of GC peak areas with a standard of n-C2, alkane. CQuantitation expressed as ng.g dry, extracted sediment -~, determined as above; - - Not detected.

2 - h y d r o x y a c i d s m a x i m i s i n g at Cz4 (880 n g . g - t ) w e r e d e t e c t e d in t h e u p p e r m o s t s e d i m e n t , b u t o c c u r r e d o n l y in t r a c e a m o u n t s in s e c t i o n 2.

Other lipid components o f sediments la and 2 A d d i t i o n a l lipids i n c l u d e d s t e r o i d a l a n d a l i p h a t i c a l c o h o l s , a n a l y s e d as p r e v i o u s l y r e p o r t e d for t h e o r g a n i s m s a n d surficial s e d i m e n t ( R o b i n s o n et al., 1984b). T h e c o m p o s i t i o n o f free a n d b o u n d n a l k a n o l s in s e c t i o n 2 is s h o w n in Fig. 5. F r e e n - a l k a n 2-ols w e r e d e t e c t e d o n l y in s e d i m e n t 2; t h e i r c o m p o s i t i o n is s h o w n in Fig. 2. B o t h free a n d b o u n d a l k a n o i s f r o m s e d i m e n t 2 c o n t a i n e d C30 a n d C32 a l k a n - l , 1 5 - d i o l s ( T a b l e 5); t h e free C3o c o n s t i t u e n t

:

2

, IJ dJIJ. I.

10

1~

i0

Carbon

Is

2o

2s

Number

Fig. 4. Distribution o f b o u n d fatty acids ( A ) 3-hydroxy and (B) (o-hydroxy in sediment 1 (upper) and 2 (lower). lso- and antebo-branching in 3-hydroxy acids is depicted as in Fig. 3. 60- Sediment 2 Free

26

pg g-~

1

16 22

Bound

40

20

15

20

25

,l,l_. I.

30 15 20 Carbon Number

25

30

Fig. 5. A b u n d a n c e s o f free (left) and b o u n d (right) n-alkanols in sediment 2.

P.A. CRANWELLet al.

518

Table 5. Alkane diols isolated from sediment 2 Abundance ( n g . g -~ ) Compound

Free

Bound

C~0 1.15 diol C~ diol isomers + keto-ol a C32 1,15 diol C32 diol isomers + keto-oP

710 590 300 200

820 ND 390 ND

reactions (e.g. hydrogenation of AS-stenols and formation of AZ-sterenes and A3'S-steradienes) were noted. The lipid components now reported for the organisms and surface sediment are indicative of gdditional sources and changes occurring during early diagenesis, as discussed in sections (a) and (b). The composition of lipid classes in sediment la, including the hydroxylic and steroidal components reported for organisms and sediment l in part I, the hydrocarbons, ketones and carboxylic acids noted above, and wax esters, are thought to reflect similar input to the present, modified by post-depositional change. In section (c) these lipid constituents of sediment 2 are used to assess the input sources and depositional environment of the lake c. 1900, based on the features noted during contemporary deposition.

(a) Lipids of Organisms in the Water Column Hydrocarbons The dominance of the n-C~7 alkane in the ciliated protozoa presumably reflects an algal or detrital diet, as n-C~: is dominant in many algae (Weete, 1976) and bacteria (Hun and Calvin, 1969). The bimodal distribution, maximising at Cl7 and C29, in Clathrochloris hypolimnica is consistent with that reported for a closely related Chlorobium species (Han and Calvin,

21

40

18

20

6 ll_ |

80

[23~ s

1 .I-

.--

17 i

2 29

h.

Sediment ia Free

I ,! a

26

,tag g~1

23 I 29

,o

40

Free

lug g - I 20

'1 Ll l ,lif

18 6

1[

al 5

26 I

I a.I

.. 10

21

15

20

--

25

2

Bound

•

. .

,3

21

II

h

Abundance (#g'g dry wt ~) Compound

°Includes LI3- and 1,14-diols and 15-one-l-ol co¢luting with 1,15-diol. ND = Not detected.

Sediment 1 t Free

Table 6. Sterenes and steroidal ketones occurring in sedimentary free lipids

!i

a

g tb 15 2b 2"s

Histogram bar number

Fig. 6. Distribution of free and bound sterols in sediments. Bound sterol in sediment la not determined. For sterol identification see Key (Table 8).

Cholest-2-cne Cholesta-3,5-diene 24-Methylcholest-2-ene 24- Methyk:holesta-3,5-diene 24- Et hylcholest-2-ene 24- Ethylcholcsta-3,5-diene 5~(H)-Cholestan-3-one 5a( H)-Cholestan-3-one 24- Methyl-Sa (H)-cholest-22-en-3-one 4a -Methyl-Sa (H)-cholestan-3-one 4a,24-Dimethyl-5,, (H)-cholest-22-¢n-3-one 24-Methyl-Sa (H)-cholestan-3-one 24-Ethyl-5/~(H)-cholestan-3-one 4a,24-Dimethyl-Sa (H)-cholestan-3-one 4:t -23,24.Trimethyl-5• (H)-cholest-22-en-3-one 24- Ethyl-5~ (H)-cholestan-3-one 4~,23,24-Trimethyl-Sa(H)-cholgstan-3-one , ,

Sediment I"

Sediment 2~

0.63 0.26 0.13 0.34 0.49 0.36 0.4 0.3 0. I 1.8 2.5 0.6 0.8 0.5 5.9 0.9 2.2

0,10 ND 0.20 ND 0.23 0.18 Tr. 0.0 I ND 0.07 0.05 Tr. 0.0 I 0.09 0.50 0.10 0.27

ND = Not detected; Tr: = Trace amount pres¢nt. ~Sterene figures previously reported with incorrect units (Robinson el al., 1984b). bBound lipids contained 5~t(H)-cholesta-3,5-dien-7-one and corresponding 24-methyl and 24.ethyl derivatives (0.12, 0.07 and 0.41 #g-g dry wt -I, respectively).

1969). The distribution and low level of n-alkanes in the rotifer sample raises the possibility of contamination, either in the lake or during handling, however distributions lacking odd-carbon predominance occur in certain bacteria (Davis, 1968; Hun and Calvin, 1969). Hopanoids, among which hop-22(29)-ene was dominant, were detected only in C. hypolimnica, consistent with their wide distribution in prokaryotes (Rohmer et al., 1984). Among other unsaturated hydrocarbons, the presence of isobotryococcene (1) in the ciliated protozoa presumably reflects the algal diet, as this compound occurs in a colonial green alga, Botryococcus braunii (Cox et al., 1973), which has been observed in Priest Pot. The absence in the ciliates of the major B. braunii constituent, botryococcene (2), may result from an algal strain producing different proportions of isomeric alkenes, physiological factors or biochemical modification of botryococcene by the ciliates. Alternatively, a related algal species may be the source, however, a culture of Dictyosphaerium pulchellum, a major colonial green alga in Priest Pot (Irish, pets. commun.), did not produce either isomer (Cranwell, unpublished). The absence of isobotryococcene in the rotifer sample may reflect the inability of organisms smaller than the major ciliate species to feed on colonial algae. Isobotryococcene was not detected in underlying sediments.

Ketones The isoprenoid 6,10,14-trimethylpentadecan-2-one, present in the rotifers and in C. hypolimnica, occurs widely in sediments, showing a stereochemistry compatible with an origin from phytol (Brooks et al.,

Lipids of aquatic organisms Table 7. Molecular composition of straight-chain esters Chain length 28

34

36

38

40

42

44

46

Alkyl-acyl

I

la

2

18-10 17-11 16-12 15-13 14-14 18-16 17-17 16-18 14-20 20--16 18-18 16-20 15-21 14-22 24-14 22-16 20-18 18-20 16-22 26-14 24-16 22-18 20-20 18-22 16-24 26-16 24-18 22-20 20-22 18-24 16-26 28-16 26-18 24-20 22-22 30-16 28-18 26-20 24-22 22-24

ND ND 53 25 22 91 ND 7 2 23 63 9 I 4 ND 47 34 II 8 12 37 23 21 7 ND 62 15 18 5 ND ND 9 80 5 6 4 41 33 14 8

ND 12 50 23 15 NA

73 14 13 ND ND 40 15 39 6 18 68 14 ND ND 5 42 25 20 9 4 33 17 37 5 4 35 14 35 9 4 3 21 49 12 18 NA

NA

ND 54 27 12 7 ND 46 23 23 4 4 NA

II 78 5 6 13 46 30 I1 ND

*Different alkyl-acylpairings of the same carbon number coeluted in GC. % molecularcompositionsweredetermined from relative proportions of (RCO2H:)'÷ fragments in mass spectra obtained by summing over the whole peak. I, la and 2 are core sectionsgiven in Table I. Major constituent in bold type. ND = Alkyl-acylpairings not detected; NA = No data for these constituents.

519

Alkenoic acids obtained from the organisms are probably endogenous products. Diagenetic removal of these labile compounds proceeds within the water column, thus, the polyunsaturated acids present in the ciliated protozoa are absent from the underlying C. hypolimnica sample (Table 4). Hydroxyacids were not detected in lipids obtained by saponification-extraction of the rotifers and ciliated protozoa and were only trace constituents of the free lipids isolated from the photosynthetic bacterial layer. H+-labile lipids of these organisms were not analysed, fl-Hydroxyacids present in H÷-labile (or bound) form have recently been recognized in complex lipids occurring solely in prokaryotes (Goossens et al., 1986).

Summary The stratification of organisms within the water column of Priest Pot constitutes an aquatic food web in which transformation of organic material is reflected by the lipid composition of successive populations at increasing depths. Including previously published data (Robinson et al., 1984a, b), these changes include (a) Selective degradation of shorterchain n-alkanes and n-aikanols, (b) Rapid breakdown of A5'7- and more highly unsaturated sterols of algal origin, (c) Rapid metabolism of polyenoic acids in the anoxic water column, (d) increasing abundance of bacterial biomarkers (hopanes, hop-22(29)-ene, and i/ai-branched acids) with increasing depth, (e) bacterial hydrogenation of A5 sterols via ketone intermediates, shown by contemporaneous increases in 5fl(H)-stanols and 5,,(H):A 5 ratios and by the formation of stanones and 3~-OH stanols in the bacterial layer.

(b) Organic Geochemistry of Contemporary Bottom Sediments Hydrocarbons

The distribution of free n-alkanes isolated from sediment 1, showing only a weak C~7, consistent with its reported breakdown in anaerobic sediments (Giger et aL, 1980), together with major C27, C.,9 and C3~ constituents (Fig. 1), suggests the majority of the sedimentary n-alkanes originate from the epicuticular wax of higher plants (Eglinton and Hamilton, 1967; Cranwell, 1973). The absence of an unresolved complex mixture (UGM) and the low concentrations of Monocarboxylic acids 17~(H),21fl(H)-hopanes (Table 2), one of which, n-Alkanoic acids with chain lengths greater than 22R-17~(H),21fl(H)- homohopane can have a bio20 have been attributed to input derived predomi- logical source (Taylor et aL, 1980), suggest that nantly from higher plants, while those in the CI2--Cj8 pollution levels are minimal. Bound n-alkane distrirange have been attributed to aquatic organisms butions peaking at C~7 and lacking odd-carbon pre(Simoneit, 1978). The distributions in the organisms dominance, as in sediment I, have been previously (Fig. 3) are consistent with this data; in addition noted in lake sediments (Cranwell, 1981b), settling C. hypolimnica shows the highest content of iso- particulate matter (Meyers et aL, 1984), and in deand anteiso-branched C~5 and C~7 fatty acids, con- cayed cyanobacteria (Cranwell, 1979). Laboratory stituents occurring widely in gram-positive eubacteria simulated decay of plant material gave a similar (Goldfine, 1982). distribution (Johnson and Calder, 1973); these minor 1978). As lipid extraction of rotifers occurred under alkaline conditions, the C~8 ketone may here be an artifact derived from intact chlorophyll (Volkman et al., 1981). The co-occurrence of higher plant triterpenoid ketones in C. hypolimnica (Table 3) suggests that here 6,10,14-trimethylpentadecan-2-one may, in part, originate from higher plant detritus.

520

P.A. CRA,~WELLet al.

sedimentary constituents may thus be of bacterial origin. The exact binding mechanism for the so-called bound lipids is uncertain, but probably involves both chemical linkages and physical trapping. Some lipids may be released from polymeric material, broken down during hydrolysis; biopolymers present in sediments include cell-wall fragments, suberin and cutin. although the last two are little affected by the acidic hydrolysis used in the present study. Hopanoids occur widely in sediments and are accepted as originating largely from bacteria (Ourisson et al., 1979). Hop-22(29)-ene, dominant in C. hypolimnica and in sediment !, as in other contemporary sediments (Rohmer et aL, 1980), thus represents direct bacterial input (Rohmer et aL, 1984). lsomerisation of hop-22(29)-ene to hop-21-ene and thence to hop-17(21)-ene occurs in sediments (Ensminger, 1977); a direct bacterial input of hop17(21)-ene is also possible (Howard, 1980). n-Alkenes in the range C~-C.,7 and showing oddchain predominance were reported in sediment from two productive lakes, Greifensee (Giger and Schaffner, 1977) and Rostherne Mere (Cardoso et al., 1983), and in potential source materials, reeds surrounding Greifensee, peat and a fern, Dryopteris dilatata, in the vicinity of Rostherne Mere. The similarity between these distributions and that of free n-alkenes in Priest Pot sediment suggests that marginal reeds and peat deposits are a probable source at this site. Ketones

The occurrence only in the sedimentary free lipids of n-alkan-2-ones similar in distribution to the corresponding n-alkanes suggests in situ microbiological oxidation of the latter, a hypothesis supported by the increase in abundance of bound alkan-2-ones and presence of alkan-2-ols in the deeper sediment (see below). Hopanoid ketones, especially those with extended side chain, present only in the sediment column (Table 3) are regarded as early-stage diagenetic products of polyhydroxy bacteriohopanes (Rohmer et al., 1980), further indication of the more extensive transformations of lipids in sediments than in the water column. Monocarboxylic acids

The distribution of free n-alkanoic acids in sediment 1 is typical of contemporary sediments from productive lakes in which aquatic biota constitute the major organic input. The similar carbon distribution of bound n-alkanoic acids (Fig. 3) probably reflects bacterial cell-wall constituents, based on the presenceof iso- and anteiso-branched C~3-C~9 fatty acids, markers of bacterial input (Brooks et al., 1977; Cranwell, 1982). The greater relative input ofiso- and anteiso-branched acids, especially i/ai-Cl~, in the saturated bound acids of sediment 1 (19% compared with 9% in free acids) reflects the larger bacterial

contribution to bound lipids, possibly still contained within viable membranes, as previously observed in other sediments and algal detritus (Brooks et aL, 1976, 1977; Cranwell. 1978, 1979). The occurrence of bound polyenoic acids may reflect a direct input of intact algae containing complex lipids, as reported in another productive lake (Cranwell, 1978), The greater abundance of bound relative to free monoenoic acids in sediment 1 is attributed to bacterial input (Gillan et al., 1983b). H ydro.~v acids

A varied range of hydroxyacid types are produced by organisms, as biochemical intermediates, cell constituents or extracellular lipids (Downing, 1961; Morris, 1970). Aliphatic 2-, 3-, and co-hydroxyacids have been reported in free and bound sedimentary lipid fractions (Eglinton et al., 1968; Boon et al., 1977; Cardoso and Eglinton, 1983); stereochemical analysis of the 2- and 3-hydroxyacids enables the sources and formation pathways to be distinguished (Cranwelk 1981a): o~-Hydroxyacids occur in both microorganisms and higher plants. In the latter they exist in a free form in cuticular waxes or in a bound form in the polymers cutin and suberin (Kolattukudy. 1980). Among free acids in section I, only 3-hydroxyacids were more than trace constituents, accordingly discussion is restricted to the sedimentary bound lipids. The 2-hydroxyacids show a distribution range (C~s-C.,6) characteristic of higher plant sources, while the composition of the more abundant 3-hydroxyacids, containing a high proportion of i/ai-branched constituents (Fig. 4), is characteristic of bacterial lipopolysaccharides (Cranwetl, 1981a; Klok, 1984); a similar distribution occurs widely in recent sediments (Cardoso and Eglinton, 1983). Lower ratios of branched/normal 3-hydroxyacids have been found in algae and cyanobacteria (Matsumoto and Nagashima, 1984). Bimodal distributions of e)-hydroxyacids, maximizing at C~o and C.,2, analogous to that in section I (Fig. 4), have been reported in surface sediment from Rostherne Mere (Cardoso and Eglinton, 1983) and Coniston Water (Robinson et al., 1987a) and in 5000 year old sediment from Esthwaite Water (Eglinton et al., 1968). As cutin and suberin resist acid hydrolysis, the method used to release bound o)-hydroxyacids from the sediment, these acids may be derived from in situ microbial transformation of fatty acids (Boon et al., 1977). The bound C:6, C:g and C30 (co-l)-hydroxyacids occurring at low levels only in sediment 1 result from (ro-I)-hydroxylation, a process known to operate in microorganisms (Miura and Fulco, 1975). Summa O"

The dominance of terrestrial input to contemporary sediment, recognised from the distribution of free n-alkanes, n-alkenes, n-alkanols, bound 2-hydroxyacids and the characteristic pentacyclic

Lipids of aquatic organisms triterpenoid ketones (Table 3), probably reflects direct autumn deposition from the deciduous marginal vegetation. Based on lipid distributions in the water column, the amount of terrestrial detritus sedimenting through the water column at the time of sampling (July) was low. Minor inputs from the latter source were evident only in the bacterial layer, e.g. n-C26 alkanol and minor amounts of olean-12-en-3one and urs-12-en-3-one. Compared with the lipids of major organisms in the aquatic food web, bacterial biomarkers and products of microbial activity are more prominent in the surficial sediment. The former include hop-22(29)ene, i/ai-branched acids and 3-hydroxy n-, i- and ai-branched carboxylic acids characteristic of bacterial cell wall lipids. Microbial activity within the sediment is inferred from the presence of n-alkan-2ones, oJ- and (oJ-l)-hydroxy acids derived from nalkanes and alkanoic acids, respectively, while hydrogenation of AS-stenols is reflected in higher values of the 5~(H):A ~ ratios in the sediment compared with the water column. Diagenetic products of bacterial lipids, including extended chain hopanoid ketones and isomerization products of hop-22(29)-ene, were also detected.

Lipid composition of sub-surficial sediment (ia) Biological monitoring of the site throughout the last two decades showed high productivity dominated by green algae (Goulder, 1972; Vincent, 1980; see also Part I), also large ciliate populations (Goulder, 1972; see also Part I) and hypolimnetic deoxygenation in summer (Gorham, 1960; Robinson et al., 1984b). Changes in lipid composition, relative to section 1, may thus be attributed to diagenetic modification of distributions noted in surficial sediment. The intermediate section la (8-14cm) gave free n-alkane and alkanol distributions showing selective loss of C,7 and Ci9 alkanes and Ci4-Ct~ alkanols, respectively, relative to the corresponding distributions in section 1, thus continuing the trend within the water column and surficial sediment and providing analogous results to those obtained in recent deposits from another productive lake (Cranwell, 1984a). The distributions of C21-C35 n-alkanes, C2~-C27 n-alkenes and C,,.,--C32n-alkanols in section la closely resembled those in the surface sediment, as also did the distribution of homologous wax esters, dominated by constituents (C3s--C52) typical of higher plants (Table 7). The similarity in molecular composition of corresponding major wax ester constituents probably reflects a similar higher plant input (Cranwell, 1982). The above data on lipid composition strongly indicates that little change in the terrestrial component of the sediment has occurred during the last two decades. The similarity in abundance of 4~-methyl- and AT-sterols relative to the more widely-occurring AS-stenols (Fig. 6) suggests a qualitative similarity in algal input, consistent with monitoring data.

521

(c) Organic Geochemistry of 80-Year-Old Sediment Priest Pot was formed about 400 years ago from a northern arm of Esthwaite Water that passed over a shallow glacial sill at its southern end. Sediments transported by an inflow were deposited in this shallow region, separating Priest Pot (Marler, 1952). The lake is now filling rapidly with fen peat; surveys show that the surface area has diminished by 25% in the last 100 years. Photographs show that the present-day willow/alder stand closely surrounding the lake margin has become denser during the last 40 years; trees were essentially absent in the first decade of this century when section 2 was deposited. There is no direct evidence about aquatic organisms or trophic status in the period 1900-1910, thus the organic input and nature of the depositional environment must be deduced from the stable biomarkers and products of early diagenesis, as recognized in the above study. The primary productivity of lakes is an important factor influencing lipid distributions in the bottom sediments (Kawamura and Ishiwatari, 1985), hence the value of organic geochemical studies.

Hydrocarbons In sediment 2, deposited c. 1900-1910, free nalkanes show a distribution (Fig. 1) in which C~, C,5 and C2~ are enhanced with respect to C3~, but show a decrease in absolute abundance, relative to contemporary deposits. Diagenesis would be expected to preferentially remove n-C23 relative to n-C31, as noted by Quirk (1978) in Sphagnum peat, thus the observed enhancement of shorter chain alkanes probably reflects source differences associated with the development of tree cover or aquatic macrophytes (Cranwell, 1984a). Free n-alkenes (Fig. 1) are less abundant than at the surface, consistent with the greater lability of unsaturated compounds (Kawamura et al., 1980; Cardoso et al., 1983), but show a chain length distribution having a lower odd-carbon predominance. The latter may reflect input of biota having lower nutrient requirements, as n-alkenes in Sphag. num peat (Quirk, 1978) and those in sediment from a lake receiving input from acidic peat (Cranwell, 1981b) show an even-carbon predominance. This suggestion is supported by the absence of carbonates in section 2, as shown by elemental analysis; the greater availability of nutrients today is evident from the luxuriant marginal vegetation. Conversion of n-alkanols into n-alkenes during isolation is discounted because of dissimilar distributions (see Fig. 5). 2,6,10-Trimethyl-7-(3-methylbutyl)-dodecane, detected only in section 2, occurs together with related monoenes and pseudohomologous C25 compounds in both marine and freshwater sediments (Yon et al., 1982; Rowland et al., 1985). The parent alkane was isolated from field samples of an alga, Enteromorpha prolifera (Rowland et al., 1985), typical of nutrientrich lowland lakes and estuaries, however the alkane also occurs in sediments of oligotrophic, upland lakes

522

P.A. CRANWELLet al.

(Cranwell, 1982; Robinson et al., 1987a), implying alternative sources having different nutrient requirements. The recognition of six ring-A degraded tetracyclic terpenoids, including des-A-lupane, only in sediment 2 is indicative of the more extensive breakdown of higher-plant input in the older sediment (Corbet et al., 1980); similarly the greater abundance and diversity of hopanes in sediment 2 (Table 3) reflects the greater bacterial contribution and increasing extent of isomerization, respectively, with increasing depth. The marked increase in abundance and the carbon distribution of bound n-alkanes (Fig. 1) in this section may reflect incorporation of higher plant alkanes into polymeric humic matter resulting from decomposition processes in situ (Khan and Schnitzel 1972). Ketones

Free alkan-2-ones in sediment 2 are accompanied by alkan-2-ols showing a similar distribution (Fig. 2) suggesting that the latter are intermediates in ketone formation by microbiological oxidation of n-alkanes, as postulated by Allen et al. (1971). Such a relationship has also been recognised in an oil shale (Chicareili et al., 1984). The distribution of bound alkan-2-ones in the range Cj9-C33 resembles that of the free aikanes of the same sample, rather than the bound alkanes. Together with the proposed in situ formation of free alkan-2-ones, this suggests that in Priest Pot bound alkan-2-ones arise by binding of free alkan-2-ones, rather than oxidation of bound alkanes. An analogous transfer of free to bound lipid components was postulated for fatty acids in a marine sediment (Shaw and Johns, 1985). The composition of pentacyclic triterpenoids in lacustrine sediments shows some correlation with higher plant input inferred from pollen analysis (Cranwell, 1984b). The triterpenoid ketone distribution in sediment 1 compares well with that of sediment whose arboreal pollen was dominated by species presently circumscribing Priest Pot. The distribution in sediment 2 somewhat resembles that of sediments derived from vegetation characteristic of more-acidic soils; similar changes in vegetation were inferred from the n-alkenes. The composition of C40, C42 and C44 wax esters (Table 7) also implies a change in higher-plant source. Steroidal derivatives

Free steroidal ketones are c. ten times less abundant in section 2 than in the surface sediment (Table 6), but C27-C:9 steroidal 3,5-dien-7-ones were detected only in the bound lipids of sediment 2. These compounds, previously noted in sedimentary bound lipids (Cranwell, 1982), are of unknown origin, but the corresponding dienol may be an intermediate in the formation of steratrienes (Gagosian and Farrington, 1978). A2-Sterenes and A~'~-steradienes are also present in lower abundance, possibly indicative

of a more oxic depositional environment than at present, based on results for marine sediments (Gagosian et al., 1980); their formation has been discussed in Part I (Robinson et al., 1984b). In common with most other contemporary sediments, 4-methylsterenes were absent (Gagosian and Farrington, 1978). The utility of sterols as indicators of inputs from organisms has been discussed (Robinson et al., 1984b). Earlier studies of the organisms and surficial sediment (Robinson et al., 1984a, b) showed that 4~t-methylsterols were markers of dinoflagellate input, that algal ALsterols were resistant to degradation and that hydrogenation of A~-stenols to 5a(H) and 5fl(H)-stanols occurred in the bacterial layer and surface sediment. The higher abundance of ,l:t-methylsterols, relative to desmethyisterols, in sediment 2 may result from a greater original dinoflagellate input or from a greater resistance to degradation of 4~t-methylsterols. Studies in the marine environment support the latter hypothesis (Gagosian et al., 1980). The increase in abundance of 4~t,24-dimethyl-5~t(H)-cholestan-3fl-ol and 4ct,23,24trimethyl-5~(H)-cholestan-3/i'-ol (Fig. 6, constituents 24 and 29) with respect to their A22-unsaturated analogues (Fig. 6, constituents 17 and 26) may reflect a change in species composition with increasing depth; however in Lake Kinneret, which has had a constant dinoflagetlate input, in terms of species composition, the increase in abundance of dinostanol with increasing sediment depth was attributed to hydrogenation of the A22-bond in dinosterol (Robinson et aL, 1986). Among the desmethylsterols, C:r components appear to be transformed more Table 8 Key to sterols in Fig. 6 Histogram bar No. I 2 3 4 5 6 7 8 9 I0 lI 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Name

Structure

5fl(H)Cholestan-3/Lol la 5~t(H)-Cholestan-3~ -oi Ilia 27-Nor-24-Methylcholesta-5,22-dien-3fl-o! Vb Cholesta-5.22-dien-3~ -ol Vc Cholest-5-en-3p-ol Va 5,,(H)-Cholestan-3p-ol Ila 24-Methylcholesta-5.22-dien-3/~-ol Ve 5a (H)-Cholest-7-en-3.O-ol Via 24-Methyl-5~,(H)-Cholest-22-en-3fl -ol lie 24- Ethyl-5,8(H)-cholest-22 -en-3.6'-oi If 24-Etbyl-5.B(H)-cholest-22-en-3~ -ol IVf 4~ -Methyl-5~(H)-cholestan-3,8 -ol VIIa 24-Methylcholest-5-en-3# -ol Vd 24- Ethyl-5~O(H)-cholestan-3fl -oI lg 24- Ethyl-5~ (H)cholestan-3,, -ol IIlg 24-Ethyl-5,8(H)-cholestan-3a -oI IVg 4a,24-Dimethyl-5,, (H)-cbolest-22-en-3/Lol Vile 24-Ethyleholesta-5,22-dien-3,8-ol Vf 24- Ethyl-5~ (H)-cholest-22-en-3/I-ol Ilf 24-Methyl-5~ (H)-cholest-7-en-3# -ol Vld 24-Ethylcholest-5-en-3/Lol Vg 24- Ethyl-5,,(H)-cholesta-7.22-dien-3fl-o[ Vlf 24-Ethyl-5~,(H)*cholestan-3#-ol Ilg 4~.24- Dimethyl-5~ ( H)-cholestan-3fl -ol Vlld 4z,23,24-Trimet hyleholesta-5,22-dien-3,6 -ol VlIlh 4a,23,24-Trirnetbyl-5~(H)-cholest-22-en-3t8 -ol Vllh 4,:~-Methyl.24-ethyl-5o,(H)-cholest-22-en-3B -ol Vllf 24- Ethyl-5~ (H)..cholest- 7..en-3p -ol VIg 4a,23,24-Trimethyt-5~(H)-cholestan-3~ -ol VIii

Lipids of aquatic organisms

I

~

II

a

d

b

e

523 TIT

/~

h

c

i

Scheme 2. Sterols listed in Table 8.

rapidly than C29 sterols, a phenomenon that has also been observed in microbial mats (Edmunds, 1982; Boudou et al., 1986). The low abundance ofA 7 sterols in sediment 2 (Fig. 6 constituents 8, 20, 22 and 28) presumably reflects a smaller contribution from certain species of the Chlorophyceae to the total algal input than in the surface sediments. Dinoflagellates, however, were prominent amongst the phytoplankton of the lake during the first decade of this century. Alkan- l-ols and alkane- 1,15-diols

The lower relative abundance of free short-chain alkan- l-ois (Fig. 5) is consistent with their more rapid degradation in sediments (Quirk, 1978; Cranweil, 1981b); the greater abundance of bound aikanols implies that formation occurs within the sediment. A similar change in distribution with increasing depth, with n-Ci6 replacing n-C u as major constituent, occurred in the sediment profile from Coniston Water (Robinson et at., 1987a).

Alkane-l,15-diols (C30 and C32) were first reported in marine sediments (De Leeuw et al., 1981; Smith et al., 1983) but have recently been detected in two lacustrine sediments (Robinson, 1984). Such compounds have been isolated from a marine cyanobacterium, Aphanizomenon flos aquae (Morris and Brassell, in press); their occurrence in lacustrine sediments suggests that freshwater cyanobacteria may also produce aikan-1,15-diols. Previously, dinoflagellates and coccolithophodds have been suggested as possible algal sources (Smith et al., 1983), but the latter are exclusively marine species. Furthermore, the principal dinoflagellates of Lake Kinneret, the first modern lacustrine source of aikan- !, 15-diols, and Priest Pot do not produce these diols (Robinson et al., 1987b), hence, cyanobacteria appear to be the most feasible source in Priest Pot. The absence of alkane-l,15-diois in sediment 1 would thus indicate a change in the cyanobacterial population of the lake, or perhaps that these compounds were released during lipid diagenesis.

524

P.A. CRANWELLet al.

Monocarboxylic acids

In sediment 2 free n-alkanoic acids show a marked decrease in abundance, the relative loss of homoIogues below C,.0 being three times greater than that of higher members, as noted previously (see Cranwell, 1982 for references). The high proportion of bound acids (> C,,0) is unusual for such recent sediment (cf. Cardoso et al., 1983; Cranwell, I978, 1981b, 1984a; Ishiwatari et al., 1980), consequently i/ai-brancbed C~3-C19 acids are only minor constituents (10%) of the bound saturated acids. Fatty acids trapped in humic acid samples consisted mainly of constituents above C:0 (Khan and Schnitzer, 1972): this source may be significant in this organic-rich sediment. No change in source of bound 3-hydroxyacids is suggested by their distribution (Fig. 4A), however the reduced abundance of Ct0 and C~, homologues may reflect the metabolizable nature of such compounds; similar changes occurred in the profile of Rostherne Mere (Cardoso and Eglinton, 1983). The increase in abundance of ~o-hydroxy acids with increasing sediment depth (Fig. 4B) implies that there has been a change in input, or, more likely, in situ formation of bound ¢n-hydroxy acids. Accumulation of hydroxycarboxylic acid polymers in a peat moss was also age-dependent (Ekman and Karunen, 1982). Summao'

The organic geochemistry of the sediment deposited e. 1900 thus reflects a qualitative change both in higher-plant input, based on the compositions of free

Depth Icm)

1

2

Ster01s 3 4 5

6

n-alkanes, n-alkenes and triterpenoid ketones, and also in input from aquatic biota, from the virtual absence of A'-sterols, previously shown to be resistant to degradation and attributed to green algae, and the presence of 2,6,10-trimethyl-7(3-methylbutyl)-dodecane, for which only one specific source has been suggested. The abundance of 4:c-methyisterols, however, suggests a significant dinoflagellate input, as at present, but the molecular composition may reflect either a different species composition or hydrogenation of the A:-"bond during burial. The more-extensive breakdown of organic matter in deeper sediments is inferred from the detection of ring-A degraded triterpenes and also from the greater abundance and diversity of bacterial hopane derivatives. For diagenetic products such as AZ-sterenes and A~'~-steradienes, lower abundances suggest a more-oxic depositional environment than at present. In situ formation of bound n-alkanes, nalkan-2-ones and co-hydroxyacids from the corresponding free lipids also occurs during the early stages of lipid diagenesis. Lipid sources and their breakdown during this century

The sources of biological markers and the products of early-stage diagenesis in the profile are summarized in Fig. 7. Primary inputs include constituents 1, 2 and 4 from algal populations in the surface waters, and constituents 5 (in part) and 7 from dinoflagellates, collected earlier in the year (Robinson et al., 1984a) from a layer 1.2 m below the water surface. Except for triterpenoid ketones (15), bioOther Hopane' Steroids ~ r i v s 7 8 9 10 11

1'2

Other I}piOs '13 14 15 16

17 18

W A T

•50 Rotifers •)50 C~(iates

E R

'380

Photosynttletlc I~a~e¢ia

" "1

0 Age (yr)

$ E D t M E N T

I,

.J

i?..,°

i

Fig. 7. Changes in abundance of lipids in water and sediment profile of Priest Pot. Key to constituents: Sterols, (I) A7, (2) A5"7,(3) As.7.2:and As'7'~m'~, (4) As, (5) 5~(H)-stanols, (6) 53(H),3B-ol, 5p(H),3-,-ol, 5~,(H),3~-ol and 5~(H) +/~(H) stanones, (7) 4~-methylsterols; Other steroids, (8) A2 and A3'ssterenes, (9) A3's steradien-7-ones (bound); Hopane deratives, (10) Hop-22(29)-ene, (l l) Isomeric hopenes, 17~(H),21fl(H)- and 17/~(H),21-,(H)-hopanes, bound C~2 hopanoic acids; Other lipids, (12) Ci4-C20 n-alkangs and alkan-l-ols, (13) Bound i/ai-branehed fatty acids, (14) Bound 3-OH earboxylic acids, (15) Higher-plant derived triterpenoid ketones, 06) Des-ring A hydrocarbon analogues of group 15, also alkan-2-ots, (17) Polyenoie carboxylic adds, (18) Conversion of free to bound n-alkanes, alkan-2-ones, oJ-hydroxyaeids. Lipid components are total lipids in eiliates and rotifers, free lipids in photosynthetic bacteria and free lipids in sediments, except where stated otherwise.

Lipids of aquatic organisms markers of higher-plants are not shown, being absent in organisms other than C. hypolimnica at the time of sampling. The detection, only in section 2, of ring-A degraded hydrocarbons (16), derivatives of the ketones (15), suggests that conversion is significant within a few decades. These hydrocarbons occur in surficial marine sediments (Corbet et aL, 1980), the low deposition rates of which give a smaller degree of temporal resolution for formation of (16). Reactions mediated by micro-organisms, leading to formation of components 6, 8, 9, 16 and 18, are mostly initiated close to the sediment-water interface, as expected from the high population of decomposers in this region. F o r m a t i o n of bound steroidal 3,5-dien-7-ones (component 9, Fig. 7), however, is exceptional and may need oxic conditions, as these ketones were detected in surficial sediment deposited less than 8 years previously in Coniston Water, which does not become anoxic (Robinson et al., 1987a). Biomarkers indicative of bacterial input (10, 1 I, 14) also become prominent at the sediment-water interface. Acknowledgements--We thank the Natural Environmental Research Council (GR3/2951 and GR3/3748) for G-GCMS facilities and for grant aid to the FBA. N.R. received a Co-operative Award from the Science and Engineering Research Council. Mrs J. Waterhouse typed the manuscript. REFERENCES

Albaiges J., Algaba J. and Grimault J. (1984) Extractable and bound neutral lipids in some lacustrine sediments. In Advances in Organic Geochemistry 1983 (Edited by Schenck P. A., De Leeuw J. W. and Lijmbach G. W. M.). Org. Geochem. 6, 223-236. Allen J. E., Forney F. W. and Markovetz A. J. (1971) Microbial subterminal oxidation of alkanes and alk-lenes. Lipids 6, 448--452. Boon J. J., De Lange F., Sehuyl P. J. W., De Leeuw J. W. and Schenck P. A. (1977) Organic geochemistry of Walvis Bay diatomaceous ooze---ll. Occurrence and significance of the hydroxy fatty acids. In Advances in Organic Geochemistry 1975 (Edited by Campos R. and Goni J.), pp. 255-272. Enadimsa, Madrid. Boudou J-P., Trichet J., Robinson N. and Brassell S. C. (1986) Profile of aliphatie hydrocarbons in a recent Polynesian microbial mat. Int. J. Environ. Anal. Chem. 26, 137-155. Brieskorn C. H. and Beck K. R. (1970) Die kohlenwasscrstoffe des blattwachses yon Rosmarinus officinalis. Phytochemistry 9, 1633-1640. Brooks P. W., Eglinton G., Gaskell S. J., McHugh D. J., Maxwell J. R. and Phiip R. P. (1976) Lipids of recent sediments, part I: Straight-chain hydrocarbons and carboxylic acids of some temperate lacustrine and subtropical lagoonal/tidal flat sediments. Chem. GeoL 18, 21-38. Brooks P. W., Eglinton G., Gaskell S. J., McHugh D. J., Maxwell J. R. and Philp R. P. (1977) Lipids of recent sediments, part II: Branched and cyclic alkanes and alkanoic acids of some temperate lacustrine and subtropical lagoonal/tidal-flat sediments. Chem. Geol. 20, 189-204.

Brooks P. W., Maxwell J. R. and Patience R. L. (1978) Stereochemical relationships between phytol and phytanic acid, dihydrophytol and C~s ketone in recent sediments. Geochim. Cosmochim. Acta 42, 1175-1180.

525

Cardoso J. N. and Eglinton G. (1983) The use of hydroxyacids as geochemical indicators. Geochim. Cosmochim. Acta 47, 723-730. Cardoso J. N., Gaskell S. J., Quirk M. M. and Eglinton G. (1983) Hydrocarbon and fatty acid distributions in Rostherne Lake sediment (England). Chem. Geol. 38, 107-128. Chicarelli M. I., Damasceno L. P. and Cardoso J. N. (1984) Diagenetic chemistry of the Paraiba Valley oil shale. In Adrances in Organic Geochemistry 1983 (Edited by Schenck P. A., De Leeuw J. W. and Lijmbach G. W. M.}. Org. Geochem. 6, 153-155. Corbet B., Albrecht P. and Ourisson G. (1980) Photochemical or photomimetic fossil triterpenoids in sediments and petroleum. J. Am. Chem. Soc. 101, 1171-I 173. Cox R. E., Burlingame A. L., Wilson D. M., Eglinton G. and Maxwell J. R. (1973) Botryococcene---a tetramethylated acyclic triterpenoid of algal origin. J. Chem. Soc. Chem. Commun. 284-285. Cranwell P. A. (1973) Chain-length distribution of nalkanes from lake sediments in relation to post-glacial environmental change. Freshwater Biol. 3, 259-265. Cranwell P. A. (1978) Extractable and bound lipid components in a freshwater sediment. Geochim. Cosmochim. Acta 42, 1523-1532. CranweU P. A. (1979) Decomposition of aquatic biota and sediment formation: bound lipids in algal detritus and lake sediments. Freshwater Biol. 9, 305-313. Cranwell P. A. (1981a) The stereochemistry of 2- and 3-hydroxy fatty acids in a recent lacustrine sediment. Geochim. Cosmochim. Acta 45, 547-552. Cranwell P. A. (1981b) Diagenesis of free and bound lipids in terrestrial detritus deposited in a lacustrine sediment. Org. Geochem. 3, 78-89. Cranwell P. A. (1982) Lipids of aquatic sediments and sedimenting particulates. Prog. Lipid Res. 21, 271-308. CranweU P. A. (1984a) Lipid geochemistry of sediments from Upton Broad, a small productive lake. Org. Geochem. 7, 25-37. Cranwell P. A. (1984b) Organic geochemistry of lacustrine sediments: triterpenoids of higher-plant origin reflecting post-glacial vegetational succession. In Lake Sediments and Environmental History (Edited by Haworth E. Y. and Lund J. W. G.), pp. 69-92. Univ. Press, Leicester, U.K. Davis J. B. (1968) Paraffinic hydrocarbons in the sulfatereducing bacterium Desulfovibrio desulfuricans. Chem. Geol. 3, 155-160. Davison W. and Finlay B. J. (1986) Ferrous iron and phototrophy as alternative sinks for sulphide in the anoxic hypolimnia of two adjacent lakes. J. Ecol. 74, 663-673. De Leeuw J. W., Rijpstra W. I. C. and Schenck P. A. (1981) The occurrence and identification of C3o, C31 and C3., alkan-l,15-diols and alkan-15-one-l-ols in Unit I and Unit II Black Sea sediments. Geochim. Cosmochim. Acta 45, 2281-2285. Downing D. T. (1961) Naturally occurring aliphatic hydroxyacids. Rev. Pure Appl. Chem. II, 196-211. Edmunds K. L. H. (1982) Organic geochemistry of lipids and carotenoids in the Solar Lake microbial mat sequence. Ph.D. thesis, Univ. Bristol, U.K. Eglinton G. and Hamilton R. J. (1967) Leaf epicuticular waxes. Science 156, 1322-1335. Eglinton G., Hunneman D. H. and Douraghi-Zadeh K. (1968) Gas chromatographic-mass spectrometric studies of long chain hydroxy aeids--II. The hydroxy acids and fatty acids of a 5000-year-old lacustrine sediment. Tetrahedron 24, 5929-5941. Ekman R. and Karunen P. (1982) Esterified long-chain hydroxy acids in green and senescent parts of the peat moss Sphagnum fuscum. Phytochemistry 21, 121-123. Ensminger A. (1977) Evolution de composes polycycliques

526

P.A. CRANV,~Lt.et al.

sedimentaires. Ph.D. thesis, Universite Louis Pasteur, Strasbourg, France. Gagosian R. B. and Farrington J. W. (1978) Sterenes in surface sediments from the southwest African shelf and slope. Geochim. Cosmochim. Acta 42, 1091-1101. Gagosian R. B., Smith S. O., Lee C., Farrington J. W. and Frew N. M. (1980) Steroid transformations in recent marine sediments. In Advances in Organic Geochemistry 1979 (Edited by Douglas A. G. and Maxwell J. R.), pp. 407-419. Pergamon Press, Oxford. Giger W. and Schaffner C. (1977) Aliphatic, olefinic, and aromatic hydrocarbons in recent sediments of a highly eutrophic lake. In Advances in Organic Geochemistry 1975 (Edited by Campos R. and Goni J.), pp. 375-390. Enadimsa, Madrid. Giger W., Schaffner C. and Wakeham S. G. (1980) Aliphatic and olefinic hydrocarbons in recent sediments of Greifensee, Switzerland. Geochim. Cosmochim. Acta 44, 119-129. Gillan F. T., Nichols P. D., Johns R. B. and Bavor H. J. (1983a) Phytol degradation by marine bacteria. Appl. Environ. Microbiol. 45, 1423-1428. Gillan F. T., Johns R. B., Verheyen T. V.. Nichols P. D., Esdaite R. J. and Bavor H. J. (1983b) Monounsaturated fatty acids as specific bacterial markers in marine sediments. In Advances in Organic Geochemistry 1981 (Edited by Bjoroy M. et al.), pp. 198-206. Wiley, Chichester. Goldfine H. (1982) Lipids of prokaryotes--structure and distribution. In Current Topics in Membranes and Transport, (Edited by Razin S. and Rottem S.), Vol. 17, pp. 1-43. Academic Press, New York. Goossens H., Rijpstra W. I. C., DiJren R. R., De Leeuw J. W. and Schenck P. A. (1986) Bacterial contribution to sedimentary organic matter; a comparative study of lipid moieties in bacteria and Recent sediments. Org. Geochem. 10, 683-696. Gorham E. (1960) Chlorophyll derivatives in surface muds from the English lakes. LimnoL Oceanogr. 5, 29-33. Goulder R. (1972) Grazing by the ciliated protozoon Loxodes magnus on the alga Scenedesmus in a eutrophic pond. Oikos 23, 109-115. Han J. and Calvin M. (1969) Hydrocarbon distribution of algae and bacteria, and microbiological activity in sediments. Proc. Nat. Acad. Sci. U.S.A. 64, 436-443. Hoffman C. F. (1984) Synthesis and geochemical applications of aromatic steroid hydrocarbons. Ph.D. thesis. Univ. Bristol. U.K. Howard D. L. (1980) Polycyclic triterpenes of the anaerobic photosynthetic bacterium Rhodomicrobium vannielli. Ph.D. thesis, Univ. California, Los Angeles. lshiwatari R., Ogura K. and Horie S. (1980) Organic geochemistry of a lacustrine sediment (Lake Haruna, Japan). Chem. Geol. 29, 261-280. Johnson R. W. and Calder J. A. (1973) Early diagenesis of fatty acids and hydrocarbons in a salt marsh environment. Geochim. Cosmochim. Acta 37, 1943-1955. Kawamura K. and Ishiwatari R. (t985) Distribution of lipid-class compounds in bottom sediments of freshwater lakes with different trophic status, in Japan. Chem. Geol. 51, 123-133. Kawamura K., lshiwatari R. and Yamazaki M. (1980) Identification of polyunsaturated fatty acids in surface lacustrine sediments. Chem. Geol. 28, 31-39. Khan S. U. and Schnitzer M. (1972) The retention of hydrophobic organic compounds by humic acid. Geochina. Cosmochim. Acta 36, 745-754. Klok J. (1984) Composition and origin of complex organic matter in recent marine sediments. Significance of bacterial biomarkers. Ph.D. thesis, Univ. Delft. The Netherlands. Kolattukudy P. E. (1980) Cutin, suberin, and waxes. In The Biochemistry o f Plants (Edited by Stumpf P. K. and Conn E. E.). Vol. 4, pp. 571--645. Academic Press, New York.

Mackereth F. J. H. (1969) A short core sampler for subaqueous deposits. Limnol. Oceanogr. 14, 145-151. Marler PI (1952) A preliminary study of the post-glacial history of the Esthwaite valley. Ph.D. thesis, Univ. London. Matsumoto G. 1. and Nagashima H. (1984) Occurrence of 3-hydroxy acids in microalgae and cyanobacteria and their geochemical significance. Geochim. Cosmochim. Acta 48, 1683-168% Meyers P. A., Leenheer M. J., Eadie B. J. and Maule S. J. (1984) Organic geochemistry of suspended and settling particulate matter in Lake Michigan. Geochira. Cosmochim. Acta 48, 442-452. Miura Y. and Fulco A. J. (1975) w-I, w-2. and oJ-3 hydroxylation of long-chain fatty acids, amides and alcohols by a soluble enzyme system from Bacillus megaterium. Biochim. Biophys. Acta 388, 305-317. Morris L. J. (1970) Mechanisms and stereochemistry in fatty acid metabolism. Biochem. J, llg, 681--693. Morris R. J. and Brassell S. C. (in press) Long chain alkanediols: Biological markers for cyanobacterial contributions to sediments. Nature. Oldfield F. and Appleby P. G. (1984) Empirical testing of 2~°Pb-dating models for lake sediments. In Lake Sediments and Environmental History (Edited by Haworth E. Y. and Lund J. W. G.), pp. 93-124. Univ. Press, Leicester, U.K. Ourisson G., Albrecht P. and Rohmer M. (1979) The hopanoids. Palaeochemistry and biochemistry of a group of natural products. Pure Appl. Chem. 51, 709-729, Prahl F. G., Eglinton G., Corner E. D. S., O'Hara S. C. M. and Forsberg T. E. V. (1984) Changes in plant lipids during passage through the gut of Calanus. J. Mar. Biol. Assoc. U.K. 64, 317-334. Quirk M. M. (1978) Lipids of peat and lake environments. Ph.D. thesis, Univ. Bristol, U.K. Robinson N. (1984) Lipids in lacustrine environments. Ph.D. thesis, Univ. Bristol, U.K. Robinson N., Eglinton G., Brassell S. C. and Cranwell P. A. (1984a) Dinoflagetlate origin for sedimentary 4~-methylsteroids and 5~(H)-stanols. Nature, Lond. 308, 439-442. Robinson N., Cranwell P. A., Finlay B. J. and Eglinton G. (1984b) Lipids of aquatic organisms as potential contributors to lacustrine sediments. Org. Geochem. 6, 143-152. Robinson N., Cranwell P. A., Eglinton G., Brassell S. C., Sharp C. L., Gophen M. and Pollingher U. (1986) Lipid geochemistry of Lake Kinneret. In Advances in Organic Geochemistry 1985 (Edited by Rullkotter J. and Leythaeuser D.). Org. Geochem. 10, 733-742. Robinson N., Cranwell P. A. and Eglinton G. (1987a) Sources of the lipids in the bottom sediments of an English oligo-mesotrophic lake. Freshwater Biol. 17, 15-33. Robinson N., Cranwell P. A., Eglinton G. and Jaworski G. H. M. (1987b) Lipids of four species of freshwater dinoflagellates. Phytochemistry 26, 411-421. Rohmer M., Bouvier-Nave P. and Ourisson G. (1984) Distribution of hopanoid triterpenes in prokaryotes. J. Gen. Microbiol. 130, 1137-1150. Rohmer M., Dastillung M. and Albrecht P. (1980) Hopanoids from C30 to C3s in recent muds. Chemical markers for bacterial activity. Naturwiss. 67, 456-458. Rowland S. J., Yon D. A., Lewis C. A. and Maxwell J. R. (1985) Occurrence of 2,6,10-trimethyl-7-(3-methylbutyl)dodecane and related hydrocarbons in the green alga Enteromorpha prolifera and sediments. Org. Geochem. 8, 2O7-213. Shaw P. M. and Johns R. B. (1985) Comparison of the lipid composition of sediments from three sites in the Venezuela Basin. Mar. Geol. 68, 205-216. Simoneit B. R. T. (1978) The organic chemistry of marine sediments. In Chemical Oceanography (Edited by Riley

Lipids of aquatic organisms J. P. and Chester R.), Vol. 7, pp. 233-311. Academic Press, New York. Smith D. J., Eglinton G. and Morris R. J. (1983) Occurrence of long-chain alkan-diols and alkan-15-one-l-ols in a Quaternary sapropel from the eastern Mediterranean. Lipids 18, 902-905. Sojak L., Krupcik J. and Janak J. (1980) Gas chromatography of all C~s--Cts linear alkenes on capillary columns with very high resolution power. J. Chromatogr. 195, 43--64. Taylor J., Wardroper A. M. K. and Maxwell J. R. (1980) Extended hopane derivatives in sediments: identification by tH NMR. Tetrahedron Lett. 21, 655-656. Vincent W. F. 0980) The physiological ecology of a Scenedesmus population in the hypolimnion of a hypertrophic pond. Br. Phycol. J. 15, 27-34.

527

Volkman J. K., Gillan F. T., Johns R. B. and Eglinton G. 0981) Sources of neutral lipids in a temperate intertidal sediment. Geochim. Cosmochira. Acta 45, 1817-1828. Wakeham S. C., Farrington J. W., Gagosian R. B., Le¢ C., De Barr H., Nigrelli G. E., Tripp B. W., Smith S. O. and Frew N. M. (1980) Organic matter fluxes from sediment traps in the equatorial Atlantic Ocean. Nature, Lond. 286, 798-800. Weete J. D. (1976) Algal and fungal waxes. In Chemistry and Biochemistry of Natural Waxes (Edited by Kolattukudy P. E.), pp. 349-418. Elsevier, Amsterdam. Yon D. A., Maxwell J. R. and Ryhack G. (1982) 2,6,10-Trimethyl-7-(3-methylbutyl)-dodecane, a novel sedimentary biological marker compound. Tetrahedron Lett. 23, 2143-2146.