4.e e
FISHERIES RESEARCH BOARD OF CANADA
Translation Series No. 2948
Higher fatty acids in fossils of mesozoic Brachiopoda
by Ch. P. lyanov, and R. Zh. StoYanova
Original title: Visshi mastni kiselini vyv vkamenelosti na mezozoiski
brakhiopodi
From: ,Godishnik na Visshva khimiko-tekhnologicileski Institut-Sofia
(Higher Institute of Industrial Chemistry, Yearbook), 15(5) :
35-45, 1968
Translated by the Translation BUreau(TP)
Multilingual Services Division
Department of the .Secretary of State of Canada
Department of the EnvironmentFisheries and Marine Service
Halifax Laboratory
Halifax, N.S.
1974
11 pages typescript
i' Vze
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TITLE IN ENGLISH - TITRE ANGLAIS
Visshi mastni kiselini vyv vkamenelosti na mezozoiski brakbiopodi
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TITRE EN LANGUE ÉTRANGÈRE (TRANSCRIRE EN CARACTÈRES ROMAINS)
Higher fatty acids in fossils of mesozoic Brachiopoda
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Godishnik na Visshya khimio-tekhnologicheski institut . - Sofia,
vol.15, No.5, 1968, p.35-4..5
Ilig,her_y acids in fossils of mesozoic.Brachiopoda.
by
Ivanov,Ch.P. and 3toyanova,R.Zh.
During the last ten years a large number of studies
in organic geochemistry were performed on the fossilst
content in organic coqipounds.
The discovery of such compounds as hydrocarbons, fatty
acids, amino acids, prrphyrins, sterols and others is assumed
to be the chelhical proof of vital processes at the time when
the given layers were formed. This assumption is true only
if the named ce.ipounds are stable and may subsist for billions
of years and if they ar -,, not simply synthetized by an inorganic
way.
r'rom another Point of view, these studies are aimed
at establi,,hin[,; the relation between the organic compomds found
in fossils (unearthed former live oràanisms) an those contained
in contemporary organisms, taking into account the chemical
Changes caused by dia?;enesis.
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2
In view of these aims, fatty acids with their stability
are the most suitable for such studies.
The presence of fatty acids in different sedimentary
rock, was proved several years ago /1/. The discovered fatty
acids were of both types, with even and odd number of carbon
atoms, the odd-numbered ones being less frequent and appearing
in lesser amounts /2/. 3tudies on the ratio between the even
and the odd-numberec: fatty acids showed that this ratio
diminishes in older foasils, i.e. thai-r content
odd-numbered
fatty acids diminishes /I/. Both types of fatty acids (with
even and odd number of carbon atoms) were found in the
Montanian too /3/.
The systems studied by Abelson showed that fatty acids
/3 6
with 14, 16 and 18 carbon atoms are the most frequent acids
contained in fossils up to 500 millions years old. It seems
that palmitic acid is the main component of old sedimentary
rocks. A proof pleading for this assumption is its presence
everywhere and in big amounts in contemporary ore2.anisms. Using
gas chromatography, Abelson found that the C 14 , C 16 and C1 8
ecids content varies frbm 2 x 10 -4 to 10-5 g (of organic
matter) /4/.
Research performed by Abelson on thermie degradation
of saturated and unsaturated fatty acids at 1900 C in absence
of oxygen showed that this temperature haslittle influence on
saturated (Cl) and monounsaturated (C18:) fatty acids /5/.
Jata were also published on the isolation from similar
fossils of fatty ecids with isostructure of the carbon chain
/6/.
.-.•
3
In our first research on fatty acids contained in
fossils, we have isolated fatty acids from 18 samples of
contemT)orary and tertiary corals of the genera Pontes Tarbellastraea end Heliastraea. Jubmittini_; the isolated fatty acids
to gas-liquid chvomabography, we studied their composition and
the content in each type of fatty acids. We found that palmitic
and stearic acids were quantitatively predominant in all
samples. The diagram representing the dependence of the natural
logarithm of the ratio micromoles per mg of nitroP,en on the
age shows a linearly decreasing trend of the amount of fatty
acids contained in the studied fossils, with the increase of
their age /7/. In order to ascertain this trend, it was necessary to study the fatty acids content of a bigger number of
samples, includinP: those from older geologic eras and periods.
To do this, the present study tries to establish the amount
of fatty acids contained in mesozoic Brachippoda and to find
out the dependence between the content of each fatty acid and
the samplest absolute geolo:ic
Material and methods
In tbis research e studied seven fossils of l'rachiopoda
whose absolute age varied from about 46 to over 200 million years.
Table 1 shows the studied fossils and some of their characteristics.
1. Preparation of samples
;,11 bteps were taken in ardor to avoid external
impurities of organic ori2.in. For this purpose, the samples
\Jere cleaned with a mebalic brush and washed in order idth
hydrochloric acid, benzol and uistilled water, • ibb exbraordL",r
4
care not to permit contamination with organic matter. The
cleaned fossils were dried at 105 ° C, crushed in fine 1Dieces
with a metalic hammer and then ground to a fine powder in
an agate mortar.
2. jeparation of crude fatty acids
°amples, 8 g each, were weighed from this powder
which was dried to constant weight. The samples were solved
in 50 ml of 6N hydrochloric acid. This solution underwent
extraction repeated 4 times with 60 ml of ether purged of
peroxyde. Thus, the fatty acids contained in fossils as
free acids, salts or esters, as well as other compounds
soluble in ether, are extracted with ether. The ether solution was washed with distilled water, dried with sodium
sulfate and centrifuved in œder to eliminate the drier.
/38
Jistilling the solvent gives a pale yellow crude residue.
3. Purification of fatty acids and obtention
of their methylic esters
The obtained crude extrct was purified through
preparatory thin-layer chromatography on silica gel with
heptane and the fatty acids were extracted with benzol.
The spots were revealed with a 0.2 solution of 2,7-dichldrfluorescein or with iodine vapour.
Beside fatty acids spots, there are those of paraffinic hydrocarbons which too were separated through extraction
on silica gel with benzol. Because of their small amounts
(less than 1/10 of the am.ount of.fatty acids) the paraffins
were not studied.
5
After elimination of benzol, the obtained residue
underwent esterification with absolute methanol and sulfuric
acid as explained in our previous paper /7/.
4. Assay of acids
The obtained methylic esters were identified by galliquid chromatography after dissolution ;n a given amount of
benzol. Gas chromatography was performed with a Perkin-Elmer
apparatus, model 7, with isothermic regime, as well as with
a 3himadzu apparatus with variable temperature. The working
conditions are given beneath each figure.
Figure 1 shows the gas-liquid chromatogram of fatty
acids from B7 (Terebratula vulgaris) with isothermic regime,
whereas figure 2 gives the chromatogram of fatty acids frm
the same sample with programmed temperature. The chromatogram for the other samples are completely analogous. There
are differences only in the height of the peaks corresponding
to each acid.
Results and discussion
Following the method described in our previous paper
h/,
We
calculated the absolute quantity of eacb fatty acid
on the basis of the chromatograms wLth isothermic regime,
namely on the basis of the peaks' area. To do 50, we prepared standards expressed in micromoles per mm2 of the peaks'
area, using known control samples. The results from each
fossil are given on table 2.
The propotional contenb of each fatty acid in each
J:::::)te of the Jtulied mixture 1:,:1-3 calculated on the bais of
L39
6
chromatograms obtained with programmed temperature. The results
are given on table 3. Using these proportional contents, we calthe absolute quantities in micromoles per mg of nitrogen
for each acid on the basis of the quantity of 01 6 acid established with the chromatograms obtained with isobhermic regime for
each sample. It was assumed that the 0 16 content is identical
on both chromatograms. The results are shown on table 4.
This time, like in our previous research /7/, the
quantities of fatty acids were expressed in micromoles so as
to assure their comprability. As explained in /7/, the quantities expressed in micromoles were related to one mg of
soluble organic nitrogen which we established in microkjeldahl
upon a solution obtained after purification in a column with
ion exchange rosin of a solution in hydrochloric acid of the
fossil sample extracted with ether.
Tables 2, 3 and 4 show that in the studied fossils
C16 (palmitic acid) and C18 (stearic acid) are in the biggest
amounts. The lower acids (C12, C1'13, 0 14, 0 15 ) as well as the
higher ones ( 0 19, 0 20 , 0 21) 0 22 , C 23 G 24' 02 5' 026 ' 02 7 )
and in ,,ample B5 tbe acids 028, 029 end C30 are in snaller
amounts. Then, vo cab notice that the quantities of even
numbered acids, of 013 :. nd C18 only, are much higher than
those of the neighbouring odd-numbered acids. For the other
acids, those from C12 to 0 15 and especially from 0 19 to 0 27,
no predominance of the even over the odd-numbered could be
noticed.
culated
7
On the chromatograms obtained with isothermic regime,
we can clearly see lower peaks right after those corresponding
to C16 : and C 1 8 : . We assume that these peaks correspond to
the unsaturated acids C16 and 318 even if their presence is
surprising in such old fossils. Koreover, in /7/ we found
that Cl: can be found in the skeleton of contemporary corals
only.
On the chromatogram obtained with isothermic regime
(polar phase), right before C18, a peak appears noted with a.
This peak could be explained by the presence of a corresponding
acid with split chain. On the chromatogram obtained witU programmed temperature (non polar phase), right before C18 a peak
appears'partially coinciding with the higher peak for C18•
We consider this 'peak correspoding to the unsaturated C18 acid.
A fact pleading for this explanation is that this peak is the
most visible in the case of B3 and B4, where the unsaturated
C18 acid is in largest amount also on chromatograms obtained
with isotl—ermic re?ime. Further, in some ,,amples, we can notice
a duplication of the peaks corresponding to some other acids
(t , C14 in B2, B3, 13 4 and 35; to 015 in B1, B3 and B4; to 020
in B3 and B5; to C22 in Bi and B5). '‘de cannot say yet if this
duplication is due to the correspoding unsaturated acids or to
those with split chain (wbich appear by non polar phase before
the corresponding normal saturated acids).
Table L. shows• that the absolute quantities of acids
following C.18 calculated in micromoles per mg of nitrogen
Un the basis of
aulount of Cl6 in each sample c2,talished
'„ •
8
on the chromatograms obtained with isothermic regime) are
curiously variable depending on the age. This variation is more
clear when we calculate the percentage of the ratio between the
quantities in moles of acids following C18 and the quantity in
moles of 016 in each sample (table 5). In B4 016 is in the
smallest amount whereas other acids are in the biggest amount.
On table 2 we can see too that the 016 and C18 content
of the studied samples of Brachiopoda fossils, 46 to 205 million
years old, decreases with age. In order to better examine this
decrease, the results concer.ling the acids present in biggest
amounts (016 and 01 8) were represented on a diagram where the
• age of fossils in million years was marked on the abscissa and
the ratio micromoles/mg of nitrogen on the ordinate. After uniting with a strirebt line the points for 01() from B1 (youngest
brachiopod) and from B7 (oldest brachiopod) we can see that all
other points are situated close to this line on both its sides.
The same image is obtained for 018. For both acids, in all
samples, the points corresponding to all other acids are very
close and within the limits of possible error. Thus, generally
the dependence J 1. ' the natural loarithm of micromoles of acid
per mg of nitrogen on the age is really linear.
This important conclusion coincides with the extreme
results obtained in determining the fatty acids content of
tèrtiary corals okrer 60 million years old (our previous research PO. As these results were obtained after studying
a limited number of samples, it will be necessary to study
Jeveral Dther fo,):311,3 in , )rder to com:irm te e3tabli_)hed
9
dependence on older fossils and on 3ample3 of other families
of fossils. This will be the aim of our further investization.
Aknowledgements to Mr.Platon Chumachenko, ph.d., and to
Mr.,java Ovcharov, researcher.
Table 1.
Ta 6 sugal
,Iassu 3a suczegnasirre
,
r
1
2
C5pa3ga
reosormJa s -.3pacT1 2, 00—
lia:(0gurge
Hepang4
i3'-ek .-.)
'3-ba .pac'r
sanecres
Jim•rec
46x10
xpega
canon
B Lobothyris Subpmnctata 9c*1oleUa,r0g0n0
3
/C) c.rpasur033,Bea0—
B Terebrstu1a , zietsni
epega
an r
79:z10 6>6
C92:10
mpa
B c Terebratula solvempislac.Ba.zum,T0edyxusceo
).
- 1 2 c .K om , liga ,rox'ervico
B4 Lcbothyris grestansis
.., O
B7 Terebre tula vulsaris 1 .. c.:,1par 0 3z1.-mga,
-- Cocpince°
mpa .
611r
gap;i7r
mpa
cusvrip 167x10 6
mpsac
asuu
,B I Terebrutula sp.
•
B 2 Ter:ebratu1a csruep
4
7
c.re6egae
8 r0.0y4ea
rpagq;imeo
!49x10 6
164x10 6
233x10 6
Fig. 1.
(Dnr
.
1
Time, min.
b
9b
Fig. 2.
anxr . 2
Table 2.
'hut aptemxre
012
0 14
0 15
0
16
0 16;
17
0 18
0 18;.
-
B1
0014 0,0 1
B2
0,006 0,012 0; 019 0,011 0,079 0,010 0,005,0,070
B
0,003 0,006 0,012 0,009 0,053 0,010 0,003 0,063 0,025
B
B
B
B
3
4
5
6
7
0,023 0,020 0,209 0,015 0,008 0,170 0,030
-
0,002 0,002 0,006 0,003 0,069 0,016 0,003 0,079 0,026
-
-
0,007 0,005 0,053 0,010 0,005 0,057 0,014
-
0,004 0,004 0,079 0,007 0,003 0,070 0,020
0,009 0,005
-
-
0,060 0,015
-
0,048 0,012
9c
Table 3.
T A'B ,11 14 1.1 A 3
clg-hpxanue Ha ouemuuTe macTHH uucenaltu,onpegeneno npH racoao-TegliocTua xpomaTorpaipm npH nporpamnpalla TeuneuTypa a apoue:!'r
cmecTa OT macTHH IcHcentuni,HnonHpalla cnee npenapaTmaHarbincocionua xpouaTorpa4=
h ua o6- _ C 14
-
16
17
0 18
0 18
C 19
C 20
C 20
C9 1
0 22
C 22
0 23
0 24
25
c
26
27
0,7
1,4 45,2
2,9 1 2,0
11,0 2,0
-
6,2
1,6
1,8
3,4
1,9
1,9
1,8
1,4
1,9
2, 7 2 , 7 1,7 1,4 1,3
5,0 29,9
2,2 3,0
17,2 4,3
-
5,0
2,6
-
4,3
3,0
3,6
3,1
2,1
2,6
1,5 22,0 11;9 8,6
15,3 3,8 3,2
5,0 ,3,7
-
7,0
3,2
3,7
2,6
2,0
2,3
1,4 1,5
0,9
1,0 14,8
6,5 9,0
13,5 4,0
6,2
7,1
-
4,8
4,1 23,7
8,3 2,2
11,0 1,6 2,5
5,5
2,2
5,9
5,8
2,2
2,8
-
7,4
2,1
5,6
1,6 5,3
7,7
4,8
2,6
1,2
3,6
4,9
5,3
-
6,2
6,4
4,7
4,3
2,5
2,2
-
B
1
B2
P
3
B
4
B
5
B
6
C.1D
' 15
0 28
0 29
0 30
2,0
5,0
2,6
e-
pa3t1Uxe
3,1
_
-
B7
-
2,8
.-
7,9 22,9 8,5 5,6
-
_
12,4 3,5 -
Table 4.
T
• KO/1H OOT/30 Ha MaCTHHTB uucenunn up.Mjur W,u3noJxeHo OT UpOgOHTHOTO HU
perum
'TeptiOH
HOMMOCTBO HA
h Ha o6paffliye
•
B2
B
3
13
B
4
5
0 I9
2
0,008
-
0,024
0,006
0,010
-
0,011
0,006
0,0083,006 0,010
0,016
7
B CbOTBOTHHTO
0 23
0,006
0,012
-
0,009
0,007
-
0,024
0,02'!
0,003 0,005 0,009
20
0 21
_
_
_
0,004
.
0,008
-
0,010
0,011
0 28
2
0,005
-
0,003
.
0,007
_
0,004
-
0 27
24
0 25
0,006
0,006
0 4 06
0,004
0,006
0,006
0,007
0,006
0,004
0,004
0,013
0,006
0,006
0,004
0,003
0,004
-
0,02°
0,026
0,019
0,018
0,008
0,014
0,010
.
0,uJa
0,003
...
0,003
0,u04
0,002
0,008
-
22
26
•
-
ompeAexenowo npu
-
..:
0,012
0,012
L),008
0,004
L.
Fig. 3.
.1 3
2
0
20
1.0
100
140
148
-- - - -
B3
83 8-
401
(Ina r
3
MOO-
_
npe)6H
0 22
B6
B
u•icenimaTa C 6
OlaIpX811110.H
ji,
100 motam.
0
30
0,003
-
-
10
Table 1. Jata on studied fossils
1. jampleS
2. Provenance
3. GeoloP:ic age
4. Period
5. Epoch
•
6. Absolute age
7. Village of Gebedzhe Paleoene - Lutetian
8. City of jhumen - Cretaceous - Senian
9. Village of Komshchina,Godechko - Cretaceous - Aptian
10. Village of Granitovo, Belogradchishko - Jurassic - Batian
11. Village of Balik, Tolbukhinsko - Jurassic - Charmoutian
12. Village of Komshchitsa, Godechko - Jurassic - 3inemourian
13. Village of Dragovishchitsa, sofia - Triassic - Anizian
1 - Time,min.
Fig.l. Gas-liquid chromatogram of methylic esters of fatty
acids separated from Terebratula vulgaris
1.kmditions: apparabu: Perkin-Elmor,model 7; flame ionization
detector; column of 200 x 0.4. cm (inner diameter); phase:',
15,; polyethylene glicol succinatc (Chromosorb
:50-0 mesh);
temperature: 185 0 0; carrier gas: nitrogen 60 ml/min.
Fig.2. Gas-liquid chromatogram of met#ylic esters of fatty
acids separated from Terebratula vulgaris
Conditions:apparatus-: Jhimadzu GC-16; flame ionization detector; column -)f 117 x 0.3 cm (inner diameter); phase:
15,3 -3E-30 (celite 100-200 mesh); programmed temperature
(40 - 3000 C; (20/rain); speed of paper: 2.5 mm/min.; carrier
gas: nitrogen (80 ml/min.).
t•111:
il
Table 2. Fatty acids content in micromoles per mg of nitrogen
established by gas-liquid chromatography with isothermic regime
Mo. of sample
Table 3. Fatty acids content established by gas-liquid
chromatography with prozrammed temperature, expressed
in of the mixture of fatty acids separated through
preparatory thin-layer chromatography
No. of sample
Table 4. Quantity of fatty acids in micromoles per mg of
nitrogen, calculated from the proportional content
and the quantity of C16 e3tablished with isothermic
reqime for each sample
No. ce sample
Fiq.3. Dependence of the quantity of palmitic and stearic acids
on the fossils' age.
:00rdinates: ordinate: natural loi;arithm of the quantity
of acid expressed in micromoles per mg of soluble organic
nitrogen; ascissa: fossils'age in millions of years.