THE FATTY ACID COMPOSITION
OF ARCTIC
PHYTOPLANKTON
A.ND ZOOPLANKTON
WITH
REFERENCE
TO MINOR ACIDS
MARINE
SPECIAL
R. W. Lewis2
Institute
of Arctic
Biology,
University
of Alaska,
College
99701
ABSTRACT
Collections of phytoplankton
and of one species of zooplankton,
the amphipod Apherusn
glucial~,, were made from the same area of the Chukchi Sea off Barrow, Alaska. The
collections
were analyzed for fatty acids, including
branched-chain
acids and positional
isomers.
The phytoplankton
contained is~ and anteiso odd-numbered
acids that have not prcviwas similar to that
ously been reported from diatoms. In other respects, the composition
of diatoms from the North Sea. The amphipods had a simpler fatty acid composition with
smaller amounts of polyenoic and branched-chain
acids than the phytoplankton.
been met because of the difficulty in obtaining reasonably pure natural phytoplankton
collections uncontaminated
by
zooplankton.
Kelly, Reiscr, and Hood
( 1959) separated phytoplankton
and zooplankton by simultaneous USCof No. 6 and
No. 20 mesh nets, and the studies by Klenk
and Ebcrhagen (1962) on the diatom, Biddulphia sinensis, and by Patton et al. (1966)
on the dinoflagellate, Gonyaulux poZyedra,
were based on collections taken during
intense phytoplankton
blooms when zooplankton tend to bc excluded (Hardy and
Gunther 1935 ) . My study reports the
fatty acid composition of collections of
phytoplankton
and one species of amphipod collected from arctic waters on successive days and in the same area during
a similar bloom.
To obtain
a detailed
analysis
of
the minor fatty acids (odd-numbered,
branched-chain,
and positional isomers of
unsaturation) , the fatty acids were collccted as carbon number families from a
SE-30 GLC
column
and rechromatographed on a DEGS column as described
by Smith and White (1966) and by Lewis
( 1967).
INTRODUCTION
Most of the information available on the
fatty acid composition of phytoplankton
has been based upon unialgal cultures
(Ackman et al. 1964; Katcs and Volcani
1966; Paschke and Wheeler 1954; Schlenk
et al. 1960; Williams 1965). Such data,
however useful, are not ncccssarily directly
related to natural food chains, since the
lipid content and composition
of algal
cultures depend on the composition of the
culture medium-principally
the amount
of available nitrogen (see Miller 1962)and on the particular growth phase of the
culture. Also, there are differences in fatty
acid composition among the various species. Therefore, collections of phytoplankton from the sea under natural conditions including several species and growth
phases should offer data more pertinent to
the study of marine food chains.
The obvious need to examine plankton
living on their natural diet has not often
1 Contribution
No. 75 from the Institute of Arctic Biology.
Drs. J. L. Barnard and T. E. Bowman
provided
information
about the amphipods,
and
Dr. T. Raeder identified
the diatoms.
Drs. L.
Irving
and F. B. Shorland reviewed
the manuscript and offered valuable
comment.
The Director and staff of the Arctic Research Laboratory
at Barrow, Alaska, assisted in the field studies,
This research was supported
by U. S. Public
Health Service Grant GM 10402,.
2 Present address: Department
of Scientific and
Industrial
Research,
Food Chemistry
Division,
P. 0. Box 8021, Wellington,
New Zealand.
MATEBIALS
AND
METHODS
Plankton collections were made among
drifting ice floes in the Chukchi Sea approximately 1.6 km offshore opposite the
Arctic Research Laboratory
at Barrow,
Alaska, on 20 September 1966. There ap35
pcarcd to be a considerable phytoplankton
bloom, for short tows (ca. 100 m) of the
NO. 20-mesh (73-p aperture)
0.25-m net
nearly filled the cod end with thick brown
sludge. Microscopic examination showed
that the collection
was predominantly
phytoplankton.
A portion of the collection
was rinsed through a Tyler No. 60 sieve
(250-p openings ) to remove the larger
forms of zooplankton; the dry weight of
this zooplankton fraction comprised 4.8%
of the dry weight of the total, Diatoms
were the major components of the phytoplankton and included six or seven species
of Chaetoceros and one or two species
each of Thalassiosira and Rhixosolenia.
These genera are described by Bursa
(1963) as pelagic and characteristic
of
the offshore waters of this area. The collections were concentrated by vacuum filtration and stored at -15C in three times
their volume of chloroform : methanol (2 : 1
v/v) to which a small quantity (ca. 0.01%)
of hydroquinone was added as antioxidant.
Amphipods are of tremendous importance in the marine fauna of Barrow
(MacGinitie 1955). A species subsequently
identified as Apherusa gZaciaZis ( Hansen)
appeared to be one of the most abundant
forms of zooplankton and was collected
with a large mesh net (ca. 1,000-p aperture) in the same area on two successive
days following the phytoplankton
collections. The animals ranged in size from 7
to 10 mm; they were sorted into homogeneous collections and placed in 20 times
of chloroform : methanol
their
volume
(2 : 1 v/v) containing hydroquinone
and
stored at -15C.
Saponification followed procedures prcviously reported ( Lewis 1967). The fatty
acids were mcthylated with boron trifluoride (Mctcalfe
and Schmitz 1961) and
were hydrogenated in methanol at atmospheric pressure over 5% palladium
on
charcoal, The GLC analyses were made
on an instrument
equipped with dual
flame ionization detectors ( F & M model
810). Analyses were made with a copper
column (5.18 m x 0.3-cm I.D.) containing
10% DEGS on 60-80-mesh acid-washed
DMCS-treated chromosorb W. Iso thermal
analyses were at 197C and programmed
analyses at 160 to 210C with a tempcrature rise of 4C/min.
Helium flow was
20 ml/min, giving a retention time of 12
min for methyl stcarate.
Carbon number fractions were collected
from a stainless steel column (0.6 m x 0.6cm I.D.) containing 20% SE-30 on the
same support as the DEGS column. The
column was operated at 197C with a
helium flow of 70 ml/min.
An effluent
splitter ( 10 : 1) was connected to a collcction port of large mass heated indirectly
by the oven. Carbon number f amilics
were collected in straight glass capillary
tubes; adcqua te amounts were collected
without the usual glass-wool plugs. The
volume of solvent normally required to
transfer these collections (ca. 1 plitcr) did
not permit analysis of fatty acids shorter
than C-14 because of the large solvent
peak. Collections of known mixtures of
stcaric and arachidonic acids were rcanalyzed on the DEGS column and gave
values within 5% of their concentration,
showing that the collection technique was
nonselective.
The C-15 and C-17 families of acids
showed tracts of 14 : 0 and 16 : 0 respectively, indicating that these major peaks
contaminated collections of the fractions
immediately
following
them. Delays in
the start of collections following standard
methyl palmitate did not affect levels of
contamination,
implicating
collection port
temperatures ( 130C) rather than timing.
Palmitoleic acid was also carried over and
markedly increased C-17 : ant&o,
whose
retention time (DEGS column) was the
same.
The fatty acids were identified by comparing the semilog plots of relative retention times versus carbon numbers for the
total spectrum and for acids of the known
carbon families. Data compiled by Ackman (1964) were used in making provisional identification of positional isomers.
A scrics of analysts of fatty acid methyl
esters of known composition
(Applied
Science mixtures K-101 and K-102) showed
FATTY
ACIDS
IN
that minor components ( ~4%) had an
error relative to the amount of the component present of less than 17% and major
components less than 12%. To include
tract acids ( <O.l% ) , the percentage compositions are listed to two decimal places.
Authentic branched-chain
acids ( Applied
Science mix No. 1) were used to establish
the performance of the two columns with
these components.
Lipids from marinc organisms generally
revcal such a complex spectrum of fatty
acids that overlapping of components, particularly on polyester columns, becomes a
serious problem. However, separation of
the total fatty acids into carbon number
and subsequent
reanalysis
of
families
these families (see Fig. 1) offers a way
of solving this problem.
Generally, the
components that were obscured or poorly
resolved in the analysis of the total fatty
acids could be determined in the analysis
of the carbon number family and could
then be related to the former by proportionality
to some other family member
clearly visible in both. The composite
peak could then be allotted to the individual components. The following
pairs
were resolved in this way: 16 : 2W4 and
18: iso, 18: 2W6 and 16:4Wl,
and 18:
3W3 and 20 : lW9. The dominance and
complexity of the C-16 acids in both the
phytoplankton
and amphipods obscured
nearly all of the C-17 acids, but 17 : 0
appeared as a shoulder poorly resolved
from the massive 16 : lW7 peak. This poor
resolution and the possible presence of
phytanic acid, which has a rctcntion time
in this range (Patton and Benson 1966)
could produce greater error in the dctcrmination of the C-17 acids than among
the C-15 acids which are seen more clearly
because of the comparative simplicity of
the C-14’s
The analysis of carbon number families
of acids, when combined with the high
resolution of the DEGS column, provided
an opportunity for a detailed examination
of minor fatty acids. The even-numbered
acids, except for those of C-14, showed
positional isomers that have been well
ARCl3.C
PLANKTON
START
FIG.
1. Gas-liquid
methyl
esters
number families
of
lected from a SE-30
DEGS. A. C-14 and
C. C-18 acids.
ac1 ‘d
chromatograms
of fatty
of phytoplankton.
Carbon
fatty acids have been colcolumn and reanalyzed
on
C-15 acids. B. C-16 acids.
documented (Ackman 1964). In Fig. 1
only the phytoplankton
analyses are illustrated; the components and proportions
were very similar in the amphipods. The
C-14 acids in both samples consistently
showed a cluster of minor components following 14 : 0 with retention times centering about 14 : 1 and a larger component
having a retention time of 14 : 2 that was
not affected by hydrogenation and whose
identity was not established. Klcnk and
38
II.
w.
LEWIS
DISCUSSION
8
9
lO:i.!co
lo:o
1l:O
12:iso
12:o
13:ko
13:o
14:iso
14:o
15:iso
15 : anteiso
15:O
15:l
16:iso
16:0
16 : lW7
16 : 2W7
16 : 2W4
16 : 3W3
16: 3W4
16 : 4W3
16 : 4Wl
17 : is0
17 : ante&o
17:O
17:l
18:k0
18:O
18: lW9
18 : lW7
18 : 2W6
18: 3W6
18 : 3W3
18 : 4W3
19:0
20 : lW9
20 : 3W6
20 : 4W6
20 : 4w3
20 : 5w3
22 : lW9
22 : 6W3
0.11
0.05
0.01
0.19
0.02
tr
0.35
0.02
0.05
0.04
9.93
0.10
0.07
0.46
0.04
0.03
15.35,
26.36
0.5,2
1.95
tr
1.30
tr
tr
tr
tr
0.05
0.01
0.03
0.01
3.391
0.03
0.01
0.261
0.06
0.02
13.40
50.30
0.24
0.913’
tr
0.44
tr
4.44
0.09
0.05
0.30
0.04
0.83
5.82
0.38
1.13
0.28
0.28
3.50
0.17
1.60
tr
0.74
0.28
16.25
1.40
5.50
0.01
0.05,
0.04
0.08
021
0.01
1.56
9.60
0.79
1.48,
0.76
1.43
tr
0.15
0.72
t-I.31
10.10
G3
Eberhagen
(1962) found a component
behaving like the Iatter in their studies
of plankton oils.
The percentage compositions shown in
Table 1 are partially confirmed by comparison of the compositions by carbon
number family from the SE-30 and DEGS
analyses when the Iatter results are pooIed
into these terms. In both samples agrecmcnt was within 3%.
Interpretation
of the results must include some estimate of the degree to
which the phytojplankton fatty acids were
carried over by diet to the amphipods.
The gut contents of A. glacialis were not
examined microscopically
and its feeding
habits must bc deduced, as far as possible,
from its morphology and natural history.
Its mouth parts arc well suited for feeding
on either diatoms or small herbivores and
it is thought to be pelagic, in contrast to
other common amphipods of arctic waters
which cling to the undersides of ice floes
and feed on fixed diatoms (J. L. Barnard,
personal communication),
This suggests
that it is an omnivore in a food chain originating in pelagic diatoms, such as those
analyzed in this study, but there is no
firm evidence of such a relationship.
Therefore, the possibility of a direct food
chain will not be considered further.
The fatty acid composition of the phytoplankton reveals the presence of iso and
ant&so odd-numbered
acids which have
not previously
been found in diatoms
( Fig. 1). It should be noted that rather
specialized
techniques
are required
to
detect these acids, particularly
at low
These acids were not
concentrations.
found in cultures of Skeletonema costatum
(Ackman ct al. 1964; Ackman and Sipos
1965), but this may have been a function
They have been
of culture conditions.
found in blue-green algae (Parker and
Leo 1965)) in ciliated protozoans ( Erwin
and Bloch 1963), and in a coccolithophore
( Ackman and Sipos 1964), suggesting a
rather common occurrence in the lower
phyla.
Iso even-numbered
acids were also
found in the diatoms I studied. Together
with the branched-chain
odd-numbered
acids, they have long been known to occur
in higher marine organisms. Thus it may
be that these acids have originated in diatoms and undergone concentration in the
food chain through prcfcrential
use of
straight-chain
acids. This hypothesis is
supported by the fact that both the total
amount of these acids in the diatoms
FATTY
ACIDS
IN
ARCI-IC
PLANKTON
39
( 0.49%) and the iso : ant&so ratio ( 2.1)
amphipods.
The pair 16 : 2W4 and 16:
were lower than those reported for other
2W7 gave ratios of 3.75 and 3.87 (Fig.
marine lipids ( 0.88-5.140/o and 3.6 to 7.7 1B). Trace amounts of 16 : 3W3 occurred
ratios, Ackman and Sipos 1964).
in both samples at about 1 : 12 compared
There is a close similarity between the with 16 : 3W4. Similarly, traces of 16 : 4W3
fatty acid compo,sitions of the pelagic di- were found in proportions of 1: 30 with
atom Biddulphia
sinensis from the North
16 : 4Wl. The massive 16 : lW7 peak obSea (Klenk and Eberhagen 1962) and the scured any minor isomers of this acid.
diatoms of this study. Larger amounts of
Among the C-18 acids ( Fig,. 1C ) , 18 :
22 : 6W3 were found in the latter, but the lW7 was found only in phytoplankton
as
other acids agree within l-2% and the a shoulder on the 18 : lW9 peak. Estimacalculated iodine value is 172 for both.
tion of quantity was necessarily approxiSince the diatoms show the same degree of mate and gave a 1 : 15 ratio with oleic
total unsaturation in their fats despite an acid. In both analyses, a minor component
environmental
temperature diffcrcnce
of followed linoleic acid and had a retention
1OC olr more, temperature
adaptation
volume appropriate for 18 : 2W4; the ratio
through increased unsaturation
does not with linoleic ( 18 : 2W6) was found to be
appear to bc occurring in arctic diatoms.
1 : 14 in phytoplankton
and 1 : 16 in the
Comparison of the above analyses with
amphipods. Identical amounts of a and y
data from the study by Ackman et al. linolenic acid were found in phytoplank(1964) on changes in the fatty acid com- ton, but the ratio in amphipods favored
position of cultures of S. costatum over the y isomer by a factor of two.
varying periods of growth shows that the
REFERENCES
best agreement is with their two-day-old
culture. This suggests that the diatoms of ACKMAN, R. G. 1964. Structural homogeneity
in unsaturated fatty acids of marine lipids.
both the arctic and the North Sea were
A review.
J. Fisheries Res. Board Can., 21:
indeed quite young and supports the idea
247-254.
that the early stages of a phytoplankton
--,
P. M. JANGAARD, R. J. HOYLE, AND I-1.
bloom permit
collection
comparatively
BROCKERHOFT. 1964. The origin of marine
fatty acids. I. Analysis of the fatty acids
free of zooplankton.
produced by the diatom Skeletonema costaThe amphipods
had generally
lower
~7~~5;.
Fisheries
Rcs. Board Can., 21:
concentrations of most fatty acids than did
.
the phytoplankton.
The total polyenoic
AND J, G. SIPOS. 1965. Isolation of the
acids fell from 38% in the phytoplankton
sa&rated
fatty acids of some marine lipids
with
particular
reference
to normal
oddto 20% in the amphipods, with a correnumbered
fatty
acids and branched-chain
sponding increase in monoenoic acids from
acids.
Comp. Biochem. Physiol.,
15: 44%
35 to 60%. By comparison, the zooplank456.
ton from the North Sea (Klenk
and BURSA, A. 19631. Phytoplankton
in coastal waters of the arctic ocean at Point Barrow,
Eberhagcn 1962) shifted in the opposite
Alaska.
Arctic, 16: 2391262.
direction-polyenoic
acids rose from 40% ERWIN,
J., AND K. BLOCH. 1963. Lipid motabin the diatom to 56% in zooplankton, with
olism of ciliated protozoa.
J. Biol. Chem.,
238 : 1618-1624.
monoencs being reduced from 30 to 16%.
Interpretation
of these findings, for the HARDY, A. C.,, AND E. R. GUNTEIER. 1935. The
plankton
of the South
Georgia
whaling
arctic material, would be difficult
withgrounds and adjacent waters, 1926-27.
Disout additional knowledge of the dietary
covery Rept., 11: 1-146.
relationships
and other idiosyncrasies of KATES, M., AND B. E. VOLCANI. 1966. Lipid
components
of diatoms.
Biochem. Biophys.
this particular crustacean.
Acta, 116: 264-278.
The proportions
of positional isomers KELLY, P. B., R. REXSER, AND D. W. HOOD.
1959. The origin of the marine polyunsatutentatively
identified
agreed in general
rated fatty acids. Composition
of some mawith previous reports (see Ackman 1964)
rine plankton.
J. Am. Oil Chem. Sot., 36
and changed little from phytoplankton
to
( 3) : 104-106.
40
n. w.
KLENK, E., AND D. EBERHAGEN.
Uber
1962.
die ungesattigten
CY-Fettsauren
des Meercsplanktons und das Vorkommen
der 6,9,12,15
Hcxadcca-tetraensaure.
Z. Physiol.
Chem.,
328 : 189-197.
LEWIS,
R. W.
1967. The fatty acid composition of some marine animals from various
depths.
J. Fisheries Res. Board Can., 24:
1101-1115.
MACGINITIE,
G. E.
1955.
Distribution
and
ecology of the marine invertebrates
of Point
Smithsonian
Inst.
Misc.
Barrow,
Alaska.
Collections,
128( 9) : l-201.
METCALFE, L. D., AND A. A. SCIEMITZ. 1961.
Rapid preparation
of fatty acid esters for
gas Chromatographic
analysis.
Anal. Chem.,
33: 363-364.
MILLER, J. D. A. 1962. Fats and steroids, p.
357-370.
In R. A. Lewin [ea.], Physiology
and biochemistry
of algae, Academic,
New
York.
Fatty
PARKER, P. L., AND R. F. LEO. 19%.
acids in blue-green
algal mat communities.
Science, 148 : 373-374.
LEWIS
1954.
PASCHKE, R. F., AND D. H. WI-IE~E~.
The unsaturated fatty acids of the alga ChZomIEa. J. Am. Oil Chem. Sot., 31: 81-85.
PATTON, S., AND A. A. BENSON. 1966. Phytol
metabolism
in the bovine.
Biochem.
Biophys. Acta, 125: 23-32.
G. FULLER, E. LOERLICH, AND A. A.
BENSON. 1966. The fatty acids of the redBioGonyaulax polyedm.
tide organism,
chcm. Biophys. Acta, 116: 577-579.
SCHLENK, H., II. K. MANGOLD, J. L. GELLEJXMAN,
W. E. LINK, R. A. MORRISSETTE, R. T.
HOLMAN, AND H. HAYES. 1960. Comparative analytical
studies of fatty acids of the
alga Chlorella
pyrenoidosa.
J. Am. Oil
Chcm. Sot., 37: 547552.
SMITII, T. H., AND H. B. WHITE.
1966. Twocolumn gas-liquid
chromatography
of fatty
acid methyl esters. J. Lipid Res., 7: 327329.
WILLIAMS,
P. M.
1965.
Fatty acids derived
from lipids of marine origin.
J. Fisheries
Rcs. Board Can., 22: 1107-1122.