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M o n o g r a p h s on océanographie methodology 1
DR F C .
VOHRA
School of
Biological
Screes
University
ot
M a l a y a
fcuala L u m p « «
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Determinat ion
o f
photosynthetic
p ig m en ts
in
sea-water
Unesco
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First
published
in
1966
by the
United Nations
Educational,
Scientific
and Cultural Organization
Place
de Fontenoy, Paris -
7
e
2 n d impression 1969
Printed
by
Imprimerie Rolland-Paris
©
Unesco 1966
Printed in France
N S .
68/XVIII. la/A
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Preface
Publication by U n e s c o of the series of
m o n o g r a p h s
on o c éa no g ra phi e
m e t h o d o
logy follows a r e c o m m e n d a t i o n
ad op te d
by the Scientific C o m m i tt ee on O c e a n ic
Research ( S C O R ) at its m e e t i ng in Halifax in 1963.
A s a
forerunner
to the series, U n e s c o
u n d e r to o k
to
distribute
to
océanographie
laboratories of the world copies of the second
a n d
revised
edition
of A
M a n u a l of
Sea-Water Analysis
by Strickland and
P a r s o n s (Fisheries R e se ar ch B o a r d
of
C a n a d a ,
1964).
T h e
series
w a s
finally
established
by the compilation a n d printing
of the present v o l u m e (No. 1). Further v o l u m e s in the series will be published
following
results
of the current
revision
of various
o c éa no g ra phi e
m e t h o d s
being
undertaken by several institutions and international bodies.
T h e
present v o l u m e
treats
various
a p p r o a c h e s
and r e c o m m e n d a ti o n s for
standardization of
determinations
of photosynthetic pigments ,
especially
chloro
phyll in phyto p l a nkto n .
Standardization
of m e t h o d s in biological
o c e a n o g r a p h y
entails
m o r e
diffi
culty
than in other fields. Regional
differences
in a b u n d a n c e and composition of
m a r i n e
c o m m u n i t i e s
call for quite a considerable
variety
of m e t h o d s w h e n one
is
mea s ur ing
standing crop and specific composition as well as productivity. T h e
m e a s u r i n g
of
p r i m ar y
productivity
directly
or
t h r o u g h
estimation of the
a m o u n t
of photosynthetic pigments in the p h y t o p l a n k t o n of a given b o d y of water is
o n e of the p r i m a r y objectives of biological o c ea n o g r a p h y .
P r o b l e m s
of standar
dization do not appear to be i ns urm o unta b l e in this respect, and standardized
m e t h o d s
for c o m p a r i s o n over a wide r an ge in space a n d season are of particular
interest.
If data for regional charts
were
c o m p a r a b l e ,
such
charts, with regional
distribution
of photosynthetic
p igm e n ts a n d their
seasonal variation in the
oce an ,
w o u l d
be m o s t helpful for the
m a p p i n g
of the
w or l d
ocean's productive areas.
T h e
storage
a n d retrieval
system for biological data, which is so urgently needed
for further progress in our attempts to unde rs ta nd the
oce an a n d
its production
of living resources,
mig ht
also
suitably
include photosynthetic
p i g m e n t
data.
T h e International C o unc i l for the Exploration of the Sea ( IC E S ) and the
U n i t ed
States National A c a d e m y of Science's C o m m i t t e e on
O c e a n o g ra p h y
h a v e
established
small
gr ou p s
of experts to consider standardization of m e t h o d s for
determination of photosynthetic
p igm e n ts
in sea-water. In D e c e m b e r 1963, the
Scientific
C o m m i tt ee
on
O ce an ic R e se arch a n d
U n e s c o
established
a Joint
G r o u p
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of Experts on Determination of Photosynthetic Pigm e n ts ( S C O R
W o r k i n g
G r o u p N o .
17).
This latter
g r o u p m e t
in Paris
f r om
4 to 6
J u n e 1964 under
Profes
sor J.
Krey' s
ch air m an sh ip
and their report is given in the first part of the present
v o l u m e .
A very important
b a c k g r o u n d
d o c u m e n t
for the
J u n e
meeting was a
survey of
existing m e t h o d s prepared
by Dr.
T .
R .
P a rs o ns
(at that time with
U n e s c o ) in his capacity as c o n v e n e r of the I C E S W o r k i n g
G r o u p
on M e t h o d s
for
M e a s u r i n g
Photosynthetic Pigm e n ts in S e a - W a t e r . D r . P a r s o n s ' survey m a k e s
u p
the second part of this
v o l u m e .
T w o Australian papers , published in the
third
a n d
fourth parts of the
v o l u m e ,
provide additional information on the m e t h o d s
a n d
their
limits.
A l t h o u g h written after the g r o u p ' s m e e t i ng , these two papers
relate closely
to its deliberations.
T h e
need
for intercomparability of
m e t h o d s
in o c e a n o g r a p h y has given
strong
i m pe tus
to
critical
analysis,
i m p r o v e m e n t
of accuracy,
simplification
of
m e t h o d s
in use and invention of
n e w m e t h o d s .
In
r e c o m m e n d i n g
that
U n e s c o
publish
these
four contributions,
S C O R
was convinced that
this
w o u l d
help to
achieve world-wide intercomparison of data, provide a reference for
intercali-
bration of other old
a n d n e w m e t h o d s ,
e n cou r age further methodological studies,
a n d
give guidance to those laboratories and
scientists w h o
w o r k in
this field.
A n y
scientific
opinions expressed in these papers are, naturally, those of
individual
scientists
or g r o u p s of scientists, and should not be interpreted as the
views of
U n e s c o .
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Contents
1
D etermination of
photosynthetic
R eport of
S C O R - U n e sc o
W o r k i n g
pigments
G r o u p
17 9
2
T h e determination of
photosynthetic
T . R . Parsons 19
pigments in sea-water. A survey of
methods
3 Comparison of the techniques used in G . F . H u m p h r e y a n d M . W o o t t o n 3 7
the determination of phytoplankton
pigments
4
Extraction
of
chlorophyll
a
from Nitzschia closterium by grinding
J.
D . K err
a n d
D .
V . S u bb a R a o
65
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1
Determination
of photosynthetic
pigments
Report
of S C O R - U n e sc o
W o r k i n g
G r o u p
17
which m et from 4 to 6 June 1964,
Unesco , Paris
A m i m e o g r a p h e d issue
of
this
report has
been
published in S ydney, Australia,
N o v e m b e r
1964
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I
Introduction
In D e c e m b e r 1963 S C O R
a n d
U n e s c o established a w o r k i n g g r o u p with the fol
lowing te r m s
of reference: to
c o m m e n t
on experimental results on the following
topics
a n d
to prepare a tentative standard m e t h o d for
p i g m e n t
determination.
1.
T y p e of
filter
for r e m o v i n g
p h y t o p l a n k to n
f r om sea-water.
2 .
Suction pressure to be applied to filter.
3 .
Necessity for grinding or sonification.
4 . Extraction
solvent.
5. Addition of basic material, e.g.
M g C C > 3
or dimethylaniline.
6.
Desiccation of
filters
before extraction.
7.
S t e a m
treatment of filters.
8 . Storage of filters.
9 .
D u r a t i o n
of extraction.
10 . R e m o v a l of extracted residue by centrifugation or filtration.
11. Precision of chlorophyll a determination at 1.0, 0.1 and 0.01 y.g
levels
u n d e r
laboratory conditions.
12.
Extinction coefficients of chlorophylls a, b a n d c.
M e m b e r s
of the
w o r k i n g g r o u p
were:
Professor J. K r e y (Kiel),
c h a i rm a n
; Professor I c h i m u r a
( T o k y o )
;
Professor K . B a n s e (Seattle); D r . S. W . Jeffrey
( Sy d ney ) ;
D r . G . F. H u m p h r e y ( S y d n e y ) ; D r . L . P.
V e r n on
( O h i o ) .
In subsequent
discussion the extra
topics listed b e l o w were
added.
13 . Optical
m e an s
of m e a s u r e m e n t : b an d -w i d t h , interference
filters,
spectro
p h o t o m e t e r type.
14. B l a n k s , corrections, equations.
15.
C o m p u t e r
cards for
p i g m e n t
data.
16. Direct determination without
solution
processes.
17. Fluorescent m e t h o d s .
18 . C h r o m a t o g r a p h i c m e t h o d s .
M o s t of these
topics
were considered in c o rre s po nde nc e
a n d
at the Paris meeting .
Statements
on s o m e of these
topics
( n u m b e r ed
differently)
are given in
this
report.
11
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D e te r mi n a t i o n of
photosynthetic pi g m e nts
in
sea-water
Dr.
Dr.
Dr.
Dr.
Dr.
G .
S.
T .
L .
C .
F.
W
R .
P.
S.
H u m p h r e y
(Sydney)
;
Jeffrey
(Sydney)
;
Parsons (Unesco,
V e r n o n (Ohio);
Paris);
Yentsch
( W o o d s
H o l e ) .
In addition to the results of
s o m e special experiments m a d e
in the laboratories
of
those present at the meeting,
three
m a i n documents w e r e
available:
Parsons,
T . R . 1963. T h e
determination
of
photosynthetic
pigments
in sea-water. A
survey
of
m e t h o d s
( m i m e o
N S / 8 9 J
issued by
U n e s c o ) .
(This survey
w a s
carried out on behalf of I C E S
Plankton
C o m m i t t e e . )
H u m p h r e y , G . F . ;
W o o t t o n , M .
1964 .
Report
to
S C O R - U n e s c o W o r k i n g G r o u p
17
:
Determination
of
photosynthetic
pigments ( m i m e o
17 60 issued
b y C S I R O ) .
(These experiments
w e r e d o n e for W o r k i n g
G r o u p
17. A paper on these
results will soon be submitted for publication.)
Ceccaldi, H .
J.; Berland, Brigitte
1964 .
Extractions par
quelques
solvants orga
niques à diverses concentrations, des pigments photosynthétiques de cultures
d e
la
diatomée Phaeodactylum tricornutum (Bohlin),
après
rétention
sur
filtres
Millipore, ou après
lyophilisation directe ( m i m e o
issued by Station
Marine d ' E n d o u m e , Marseille).
Invitations to attend the Paris
meeting w e r e
issued to international organizations
and to national committees for oceanic research. Present at the
meeting w e r e :
Professor J. K r e y
(Kiel),
chairman
;
Professor
K . B a n s e (Seattle),
rapporteur;
D r .
H .
J. Ceccaldi (Marseille);
Dr. V . K. Hansen
(IOBC,
Ernakulam);
II
Report
Preamble
Chlorophyll a, b and c concentrations in sea-water samples are used to estimate
the biomass and the photosynthetic capacity of phytoplankton. Ratios between
various plant
pigments
possibly
indicate
the
taxonomic
composition
or the
physio
logical state
of the
c o m m u n i ty .
T h e
following recommendations aim at obtaining precise measurements of
chlorophyll
a, b
and
c
in
phytoplankton.
Th e accuracy of such measurements
cannot
yet be stated since the recovery of k n o w n
a m o u n t s
of chlorophyll added
to the plankton cannot be studied. Freeze-drying
(lyophilisation)
might
be
the
m o s t
accurate
w a y
to prepare
phytoplankton
material for pigment measurements,
and should
be
used
in the
future
to evaluate modifications of pigment
extraction
m e t h o d s .
1
Type
of
filters
for removing phytoplankton from
sea-water
Evidence w a s
presented
showing that
neither
paper n o r
glass-fibre
filters
retain
all particulate matter containing chlorophyll, although
their efficiency
can
be
raised b y
covering
t h e m with p o w d e r e d M g C O s .
It
is
rcoeaoaended
that:
Phytoplankton
be concentrated by
filtering
sea-water
samples
t h r o u g h
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Report of
S C O R - U n e s c o
Working Group 17
cellulose or
cellulose-derivative
m e m b r a n e filters of 0 .45 to 0 .65 ( i pore size.
Before filtration, th e
filters
should be covered with
sufficient finely
p o w d e r e d
M g C O s to
give
a b o u t 1 0 m g / c m
2
o f
filter
area. W h e n th e
filters specified
a b o v e clog with the particular w ate r sam p l e s u se d , p ap e r filters N o . 57 5 o f
Schleicher S chüll or equivalent, covered with M g C O s as
a b o v e , m a y
b e
u s e d . I n a n y case, p or e
sizes
o f
filters
should be recorded
w h e n
reporting the
data .
2 Suction pressure to be applied to filter
W e k n o w
of no evidence
that
h i g h
suction
pressure during
filtration affects
chlorophyll
retention
a n d detection. Since nitration is not materially hastened
with the
r e c o m m e n d e d filters
b y e m p l o y i n g full
v a c u u m ,
it is
r e c o m m e n d e d that:
A
suction
o f 2 / 3 a t m b e u s e d .
3
Necessity
fo r
grinding
or
sonification
D a t a w e r e
presented showing
that
grinding the
filters
con ta in in g p h y top l an k ton
increases th e a m o u n t of p ig m en t recovered a n d reduces the t im e n eeded for
extraction. It is r e c o m m e n d e d
that:
Filters w ith th e p l an k ton b e g r o u n d for 1 m i n w i th a pestle
rotating
at ab ou t
50 0
r . p . m . in the presence of the
solvent.
It is further r e c o m m e n d ed
that:
Since i t appears
that ultrasonic
tre atm e n t o f ab o u t 1
M h z
(1,000 kc ) reduces
th e t im e n e e d e d for p igm e n t
extraction
f r o m m a r i n e
plankton , exper iments
s h o u l d b e m a d e to c o m p a r e th e effectiveness of grinding with
that
o f ultra
sonic
destruction
o f cells in regard to recovery of plant p i g m e n t s
f r o m
natural p lankton .
4
Extraction solvent
A l t h o u g h
m e t h a n o l is very efficient in
extracting
p i g m e n t from p h y t o p l a n k t o n ,
9 0 p er cent acetone is favoured at present because (a)
p ur e
chlorophyll a is m o r e
stable
in it; (b) the
chlorophyll
absorpt ion
b a n d
in the red is sharper in it; and
(c) the extinction coefficient is higher in it. Therefore it is
r e c o m m e n d ed that:
N i n e t y p e r
cent
acetone be used for
extraction. M e t h a n o l
as a
solvent
for
p l a n k t o n p i g m e n t s s h o u l d b e further investigated in view of its
superiority
over acetone with
Scenedesmus,
a fresh-water green alga difficult to
extract.
5 Addition o f basic material, e.g. M g C O a or dimethylaniline du r i n g
extraction
A d d i t i o n o f M g C O s as r e c o m m e n d e d u n d e r
Section
1 p r o m o t es
effective
filtration
a n d
facilitates centrifugation. Its presence m ig h t prevent acidification of the
extract
a n d th u s
retard
th e for m at ion o f
p h e o p h y t in .
Solutions
o f
dimethylaniline b e c o m e b r o w n u p o n
stan d in g an d m igh t cau se
a n erroneously high extinction. T h e usefulness o f adding dimethylaniline or other
basic substances to the
solvent,
to prevent
possible b r e a k d o w n
o f plant p i g m e n t s ,
s h o u l d b e
investigated.
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D e te r mi n a t i o n of photosynthetic pi g m e nts in sea-water
6
Desiccation of filters before
extraction
W e
k n o w of no evidence s h o w i n g that desiccation of
filters
s necessary before
extraction of p i g m e n t . H o w e v e r , since salt
affects
the solubility of
Millipore
filters,
filters
sh ou l d
be
s u c k e d
as dry as possible
after
filtration.
7
Steam
treatment of filters
T h e r e is no evidence that ste a m treatment is necessary to stabilize p i g m e n ts . Since
heat facilitates isomerization and oxidation of chlorophyll, the over-all effect of
steam treatment m i g h t be h a r m f u l . If it is
used,
its effect should be investigated
a n d the results stated.
8
Storage of
filters
T h e r e are data s h o w i n g that dry
filters
containing M g C O s can be stored in the
dark at +
I
o
C
or
less
for
tw o
m o n t h s w ith ou t
significant
loss of p i g m e n t (the loss
is
p r o b a b l y less t h a n 15 per cent).
H igh e s t results are
obtained by
extracting d a m p ,
u n stor e d filters.
E x p e r i m e n ts
on very long-term (several m o n t h s ) storage of filters are desired.
9
Durat ion of extraction
U n d e r
Section 3, grinding of
pl a nkt o n
before extraction is
r e c o m m e n d e d .
Te n
minutes
of s ubs equent standing m i g h t suffice for o p t i m a l extraction. Since dif
ferent types of
p h y t o p l a n k t o n
m i g h t react differently
a n d
conditions of
mechanical
destruction m i g h t be critical, it is
r e c o m m e n d e d
that:
T h e investigator sh ou l d
m a k e
checks as to the length of extraction required
after grinding the p lan k t on .
A l t h o u g h extracts ca n be stored for several
h o ur s
at r o o m
temperature
in the dark
w ith ou t
significant
loss of p i g m e n t , it is r e c o m m e n d e d that
:
Extracts should not be stored overnight.
10 Removal of extract residue by
centrifugation
or
filtration
O n the basis of evidence available it is r e c o m m e n d e d that :
Extracts
sh ou l d be cleared by centrifugation;
p r o b a b l y
10
m i n u t es
at
4 , 0 0 0
to 5 , 0 0 0
g
in a
s w i n g-ou t
centrifuge are needed.
11 Precision of chlorophyll a
determination
at 1.0, 0.1 and 0.01 ng levels
under
laboratory conditions
W e
are not in a position to specify the precision of chlorophyll
determ inations
o n m a r i n e
p h y t o p l a n k t o n
u n d e r laboratory conditions. It is unlikely that the
precision with the present procedure
will
be such that differences of 0.05 ¡xg/1
will be significant w h e n c o m p a r i n g oceanic
sa m p l es ,
i.e. in the
r a n g e
0 to 1 (j.g/1.
T h i s holds also for chlorophyll b and c. It is r e c o m m e n d e d that precision be
determined by each analyst.
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Report of S C O R - U n es co Working
G r o up
17
12
Optical means of measurement
—
b a n d width
—
interference filters
—
spectro
photometer
type
It is
r e c o m m e n d e d
that:
M e a s u r e m e n t s
be
m a d e
with
spectrophotometers
with a
b a n d
width
of at
m o s t 2 to 3 m(i, allowing
extinctions
to be read to ± 0 . 0 0 1 units.
T h e w a v e
length setting should be calibrated frequently.
If only chlorophyll
a
is determined,
instruments
with interference
filters
with not
m o r e
tha n
5 to 10
m ¡x half-band width m a y
be
us e d; w o r k i n g extinction
values
m u s t
be
determined
for
each filter
with
k n o w n a m o u n t s
of chlorophyll
a.
13 Extinction
coefficients
of chlorophyll
a, b
and
c
T h e
only available values of
extinction
coefficients for
crystalline
chlorophyll
a
in 90 per cent acetone were f r om preparations w h o s e coefficients at the p e a k in
the
red in ether were at
least
10 per cent l o w e r
tha n
the highest values in the
literature; these higher values
were
not
b a s e d
on crystalline
p i g m e n t .
It was
decided
to use the m e a n
(99.87
1/g
c m )
of the three highest available values
in ether
w h i c h
are
ba s e d
on
different m e t h o d s
of
determining
the
p i g m e n t
c o n
centration, 102.1 (Zscheile and C o m a r , 1941) , 100.9 ( Sm i th and Benitez, 1955)
a n d 9 6 . 6 (Strain et al., 1963 ) .
T h e
value 99.8 7 1/g c m (ether) was used to deter
m i n e the purity (86.81 per cent) of the
crystals
us ed by Jeffrey (unpublished) in
obtaining
absorption curves in ether and 90 per cent acetone.
F r o m
this
w o r k
of
Jeffrey the values given
b e l o w
for 90 per cent acetone
w e r e
calculated. It is
r e c o m m e n d e d that:
A n extinction
coefficient of 89 . 31 1/g c m at the m a x i m u m of
extinction
at
663
m u is used for chlorophyll
a
in 90 per cent acetone.
T h e w o r k i n g g r o u p
will ask
D r . H .
Strain to
pre pa re
an
extinction curve
of chlo
rophyll a
in acetone f r o m the
ultra-violet t h r o u g h
the
visible range into
the
infra-red.
T o
prepare trichromatic
equations,
the
extinction
coefficients of
chlorophyll
a
in
9 0
per
cent
acetone
at
645
a n d
6 3 0
m(i
ha v e
b e e n
calculated as
19.32
a n d
14.40
1/g c m .
T h e
data
for chlorophyll
b were
treated
similarly,
a m e a n value of
6 0 . 2
1/g
c m
(ether) being used to s h o w that Jeffrey's crystals were 9 4 . 1 9 per cent p u r e . It is
r e c o m m e n d e d that:
A n extinction
coefficient of 52.14 1/g c m at the m a x i m u m of
extinction
at 645
m | j .
is us ed for chlorophyll
b
in 90 per cent acetone.
T o prepare
trichromatic equations, the
extinction
coefficients of chlorophyll
b
in 90 per cent acetone at 663 a n d 630 my. h a v e b e e n calculated as 9.57 a n d 15.22
1/g
c m .
T h e extinction
coefficient for chlorophyll
c
at the p e a k in the red in 90 per
cent acetone has
been determined
by Jeffrey ( 1 9 6 3 ) . It is r e c o m m e n d e d that:
A n extinction
coefficient of
19.44
1/g
c m
at the
m a x i m u m
of
extinction
at
6 3 0 mp.
is
used
for chlorophyll c.
T o prepare trichromatic equations, the
extinction
coefficients of chlorophyll c
15
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Determination of
photosynthetic pigments
in sea-water
in 90 per
cent
acetone at 663 and 645
m ¡ ¿ h a v e
been
calculated
as 0.47 and 3.48
1/g c m .
1 4
Blanks,
correction
and
equations
O n the basis of the available evidence it is
r e c o m m e n d e d
that:
Bl an k s be 9 0 per
cent
acetone. R e a d i n g s should be taken at 7 5 0 my. to correct
for
turbidity of
the
extract
and
m u s t
not exceed 0.005 per centimetre
of
light path. This reading should be subtracted
from the
readings
at
663,
6 4 5 and 630 m | x .
T h e following trichromatic equations are
r e c o m m e n d e d :
chl.
a =
11.64
eses
— 2.16 ee45
+
0.10
eeso
chl.
b =
— 3.94 e
66
3
+ 2 0 . 9 7
e
64
5 — 3.66 e
6
3o
chl.
c
=
— 5.53
ee63
— 14.81 ee45
+
54.22
e«so
w h e r e
chl.
a {b
or
c) is
in (¿g/ml.
eees,
ee45 and
eeso
are the
extinctions
(optical
densities, absorbances)/cm
of
light path
at
663, 645, and 630 m ^
after
subtracting
the 750 m u , reading.
T h e
equations ha v e been checked
on
mixtures
of
solutions with
k n o w n
a m o u n t s
of the
three
chlorophylls.
The
results
are
s h o w n b e l o w :
Found
(ug/ml)
6.03
0.79
0.41
Chlorophyll
Added
(ug/ml)
5.87
0.74
0.37
a
Recovery
C A
103
106
110
Found
(l¿g/ml)
1.08
0.53
0.30
Chlorophyll 6
Added
(lig/ml)
0.79
0.49
0.24
Recovery
Ill
108
125
Found
(Ug/ml)
4.87
1.22
0.75
Chlorophyll
Added
(lig/ml)
5.07
1.27
0.64
c
Recovery
(
96
97
117
REFERENCES
J E F F R E Y , S. W . 1963. Purification and
properties
of chlorophyll
c
from Sargassum flavicans.
Biochem. J.,
86 : 313-18.
S M I T H , H .
C ;
B E N T T E Z ,
A .
1955.
In:
K .
Paech and
M .
Tracey
(eds.).
Modern
methods of
plant
analysis, vol. 4 , p. 142-96. Heidelberg,
Springer
Verlag.
S T R A I N , H . H . ; T H O M A S M a r y R . ; K A T Z J. J. 1963. Spectral absorption properties of ordinary
and fully deuteriated
chlorophylls
a and
b. Biochim. Biophys. Acta,
75 : 306-11.
Z S C H E I L E , F. P. Jr.;
C O M A R
C . L . 1941. Influence of preparative
procedure
on the purity of
chlorophyll components as s h o w n by absorption spectra.
Bot. Gaz.,
102 : 463-81.
Ill
Tentative standard m e t h o d for determination
of
chlorophylls
in samples of sea-water
Concentration of
s a mpl e
U s e a v o lu m e
1
of
sea-water
w hic h contains
about
1 jig
chlorophyll
a. Filter
2
thro ug h a
filter
3
covered by a layer of M g C O s .
4
16
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Report
of
S C O R - U n e s c o
W o rkin g Gro up 1 7
Storage
T h e
filter c a n b e stored in the d a r k o v er silica gel at 1 °C or less for two m o n t h s
but it is preferable to extract th e d a m p filter m m e di at el y a n d m a k e th e spectro-
p h o t o met r i c m e a s u r e m e n t without delay.
Extraction
F o ld th e filter (plankton inside) a n d
place
it in a sm all (5 to 15 m l) glass, pestle-
type, h o m o g e n i z e r .
A d d
2 to 3 m l 9 0 pe r cent a cet o n e .
G r i n d
1 m i n u t e at a b o u t
500 r .p .m . Transfer to a
centrifuge
t u b e a n d w a s h th e pestle a n d h o m o g e n i z e r
2 or 3 t im es with 90 per cent acetone so
that
the total v o lu m e is 5 to 10 m l .
K e e p
10
m i n in th e dark at r o o m temperature. Centri fuge
5
f or 1 0 m i n a t 4 ,0 00 to
5 , 0 0 0
g.
8
Carefully
p o u r
into
a gr adu ated tube so the
precipitate
is not disturbed
and if necessary dilute
7
to a convenient v o l u m e .
8
Measurement
U s e
a spectroph otom eter with a b an d -w i d t h o f 3 m y. o r
less,
a n d cells with a light-
pa th of 4 to 10 c m . * R e a d the
extinction
(optical density, a b s o r b a n c e ) a t 7 5 0 ,
1 0
663, 6 4 5 , a n d 6 3 0 m¡A
against
a 9 0 p e r cent a cet o n e b la n k .
Calculation
Subtract the
extinction
at 750 m jx f r o m the
extinctions
at 6 6 3 , 6 4 5 , a n d 6 3 0 m ( x.
D ivide the a nsw ers b y the
light-path
in centimetres of the cells. If
these
corrected
extinctions are eses, ee45, an d ee3o the concentrations o f
chlorophylls
in the 9 0 per
cent acetone extract as (¿g/ml are given by the following equat ions:
chl. a = 1 1 .6 4
eees
— 2.16 ee45 + 0.1 0 eeso
chl. b = — 3 . 9 4
eses
+ 2 0 . 9 7 e
6 4
5 — 3 . 6 6 e
6
3 0
chl. c = — 5.53 eee3 — 14.81 ee45 + 54.22 eeao
If the
values
are
multiplied
b y the
v o l u m e
of the
extract
in
millilitres
a n d
divided
by th e v o l u m e of the sea-water sa m p le in litres, the concentration of the chloro
phylls
in the sea-water is obtained as (xg/1 (=
m g / m
3
) .
N O T E S
1 T h e
a m o u n t o f chlorophyll a should be less than 10 ug , o therwise a second extraction with 9 0 per
cent
acetone
m i g h t be necessary. W i t h o c e a n w a ter a b o u t 4 to 3 litres o f s a m p l e shou ld be used; with
coastal
and bay waters,
s o m e t i m e s
one-tenth of
this a m o u n t
is
sufficient.
2
U s e n o m o r e than two-thirds of full v a c u u m .
3
Satisfactory
filters
nclude
p aper (Albet),
cellulose
(Celia
' g ro b ' ) ,
a n d
cellulose
ester
(0.43 to 0 .63 n pore
size);
th e filter sh o u ld b e 30 to 60
m m
diam eter. If
these filters
clog with inorganic detritus, use
Schleicher
Schull
373.
4 A d d
a b o u t 1 0
m g
M g C O a / c m
s
filter surface, either a s a p o w d e r or as a suspension in
filtered
sea-water.
3
A sw i n g -o u t centrifuge gives better separation than an angle centrifuge.
6
If a stoppered, graduated centrifuge tube is used, the extract c a n b e
m a d e
u p to v o l u m e and the supernatant
carefully
p o u r e d
or pipetted into the spectrophotom eter cell.
7 If turbid, try to clear b y a ddi n g a
little
100 p e r
cent
acetone or
distilled
water or by
re-centrifuging.
8 T h i s de p e n ds on the spectrophotometer
cell
u se d . T he v o l u m e sh o u ld b e re ad to 0 .1 m l .
9 Dilute
with 90 per
cent
acetone if the extinction is bigger than 0.8.
10
If the 730 (i reading is
greater
than 0 . 0 0 5 / c m light-path, reduce the turbidity as in
N o t e
7 .
17
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Det er m i nat i on of
photosynthetic
p i g me n ts in
sea-water
I V
Important subjects not discussed fully
at the working group
Accuracy
of
the equations
1. Permissible
ratios
of pigments.
2 . Permissible range of pigment concentration.
3 . Accuracy of the equations using pure chlorophyll solutions.
W o r k
on these is being carried out by H u m p h r e y and Jeffrey.
Chromatographic methods
F o r the
determination
of the
exact pigment composition
of the sample, use
should be m a d e of available chromatographic methods for pigment separations:
(a) small
columns
(Parsons);
(b)
thin
layer
methods
( M a d g w i c k ) ;
(c)
paper
(Jeffrey).
These
methods
have been adapted to
pigment analyses
in
cultures where
large quantities
of
pigments
are
present, and
to
sea-water samples with
m u c h
lower concentrations
of pigments.
For
quantitative
analyses, pigments
m a y
be
eluted and analysed with an
8 0
per cent recovery.
A paper chromatographic m e t h o d has
been
used at sea; it is an adaptation
of
the
'chromatobox'.
Samples can be run
even
in the m o s t violent
storm,
since
it is not necessary to
have
a stable horizontal base for the development of the
solvent
front.
Determination of
carotenoids
Things
which
need to
be done
:
1. Search for a
g o o d simple m e t h o d
of
separating chlorophylls
as a group
from
carotenoids
as a group in marine phytoplankton, so that a
simple
measure
of
total
carotenoids
m a y
be
m a d e .
2 . Accurate extinction values
for
fucoxanthin and peridinin.
3 . A
general extinction value
for total
carotenoids
in marine
algae.
Units
A plea is
m a d e
to oceanographers to use w a y s of expressing and evaluating data
that
have
meaning to the plant physiologist, so that important data obtained
with cultures
m a y have
application
w h e n
trying to explain
a n d
understand results
of
field experiments. For example, to express photosynthetic rates in the ocean
as
[x moles or ¡x litres C 0 2 / m g chlorophyll a + c, instead of counts C
1 4
/ m
3
,
which has no meaning in w o r k with cultures.
Computer cards
Consideration of this should be left to W o r k in g G r o u p 18: Biological Data.
S u c h consideration w o u l d be helped if a m e m b e r of
W G
17 participated in W G 1 8 .
Direct determination
Fluorescent
methods
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T h e
determination of
photosynthetic pigments in sea-water
A survey
of
methods
T . R . Parsons
Office of
Oceanography,
Unesco, place de Fontenoy,
Paris-7
1
', France
Present address:
Pacific
Océanographie
Group,
Nanaimo, B . C . ,
Canada
Manuscript
received 2 Ma y 1963
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Preface
Acknowledgement
A t
the
1962 meeting
of the International
Counci l for the Exploration of the Sea ( I C E S ) ,
the Plan kton
C o m m i t t ee
appointed convenors
for four small
working g ro u p s
to study current
m e t h o d s in biological
oceanography.
O n e of
these g r o u p s wa s to stu dy current m e t h o d s for
the
measurement
of photosynthetic pigments
in sea-water.
A s c o n v e n o r
for this
g r o u p
I
have
considered
that
s o m e preparation is neces
sary in order to provide a working group
with material with which to discuss the
problem
a n d
eventually
decide on
a
standard
procedure. I anticipated, therefore, asking per
sons to take part in a meeting of a working
group
on this subject
s o m e
time
in
1964
w hen
the preparatory w o r k h a d
been
completed.
In organizing the first part of the pre
paratory w ork as described in the following
presentation
I
a m
grateful to
those persons
w h o
returned the
U n e s c o
questionnaire
N S / 9 / 1 1 4 / 8 9
and to the persons whose
c o m m e n t s on the
o rganization
of this work
are reported
in A p p e n d i x II. Pending the
acceptance of this report by the I C E S , D r .
G .
F . H u m p h r e y ,
C S I R O ,
Cronulla, Australia,
has
agreed
to
supervise
the
type
of
experi
ments env isaged -in the report.
T h e author wishes
to acknowledge the assis
tance of D r . W . S .
W o o s te r in
the preparation
o f
this
report.
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I Introduction
U n d e r the
resolution
adopted at the
fiftieth
statutory meeting of the
I C E S
( C . Res.
1962/4(8)), the w o r k on the determination of photosynthetic pigments in sea-
water to be
carried
out m a y be
s u m m a r i z e d
as follows:
1.
A review of m e t h o d s normally used.
2. An experimental examination of various procedural steps,
leading
to
a n eventual r e c o m m e n d a t i o n for a standard procedure for pigment
analysis.
T h e following discussion
pertains
principally to the requirements in 1
a b o v e .
In addition, h o w e v e r ,
a n
attempt has been m a d e to identify the p r o b l e m s requiring
further
experimental
w o r k .
Further, it has been
a s s u m e d
that
the
m o s t immed ia t e
need is for a standard m e t h o d to be used by oceanographers m a k i n g
synoptic
surveys of large areas of o c e a n . In order to obtain c o m p a r a b l e results
from
ships
operating in different areas or at different times it is necessary to have a
reliable
universal
procedure. Particular attention has been given, therefore, to procedures
which are usable aboard ship, although they
m a y
not be so
satisfactory
for
s o m e
purpo s e s as m o r e sophisticated
m e th o d s w h i c h
could be
e m p l o y e d ,
for
e xam p le ,
in a laboratory concerned with studies on phytoplankton cultures.
II M e t h o d s currently e m p l o y e d for the determination of
photosynthetic pigments in sea-water
It is general experience
that
no
analytical m e th o d ,
h o w e v e r well described, will
b e
performed in
exactly
the s a m e w a y by different analysts.
Differences
in tech
nique w h i c h appear to be small
m a y
lead to significant differences in accuracy
a n d precision of the m e a s u r e m e n t . T h u s the following
presentation places
m o r e
e m p h a s i s on
evaluating
differences in individual techniques than on a review of
the techniques themselves.
T w o basic techniques have been e m p l o y e d ; extractive spectrophotometry
a n d extractivefluorimetry.
E x a m p l e s
of the former technique as used by
m ar i n e
scientists are given by K r e y (1939), and Richards with T h o m p s o n (1952), and
of the
latter
technique, by Kalle (1951), and Yentsch and
M e n z el
(1963). In ad
dition, reviews on these and other techniques have been
written
(e.g. K r e y , 1958;
21
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Determination of photosynthetic pi gmen ts in sea-water
Strickland, 1960) . Since m o s t workers e m p l o y the spectrophotometric m e t h o d ,
c o m m e n t
on the fluorometric
m e t h o d
is limited to the
last
portion of this report.
I nfo rm a t i o n on present m e t h o d o l o g y was obtained by sending forty-four
questionnaires to representative
m a ri n e
scientists
in twelve countries. A
s u m m a r y
o f
s o m e
thirty replies
is given in A p p e n di x I. For the m o s t part the various diffe
rences
in technique represent individual modifications of one or two m e t h o d s
—thus
it s e e m s desirable to
establish
which steps in the
procedure
are
m o s t
sensitive to s uc h modifications.
It should be noted that in
s o m e
cases the only p i g m e n t being
determined
is chlorophyll a. Since the reported precision a n d accuracy for the determination
o f
other p i g m e n t s is lower
t h a n
that for chlorophyll
a
( R i cha rds with T h o m p s o n ,
1 9 5 3 ;
Strickland and P a r s o n s ,
1960),
and
because
chlorophyll
a
is the m o s t
widely
used
p i g m e n t
for the estimation of standing
crop
or photosynthetic rate,
the following section is devoted to a consideration of p r o b l e m s
relating
to the
establishment of a standard
m e t h o d
for chlorophyll
a
alone (at the end of the
section,
there is a suggestion for the determination of other pigments ) .
T h e
s u m m a r y of data w h i c h has b e e n presented in
A p p e n d i x
I s h o w s the
a m o u n t
of variation which has been introduced
into
the stepwise
procedure
for
chlorophyll a analysis. N o indication is given of
which
individual
variations
are
m o s t c o m m o n l y
e m p l o y e d
and this has
been
omitted for two reasons. Firstly
the m o s t
p o p u l a r
use of a piece of
a ppa ra t us
or pro c ed ure tends to be biased
t ow ar d s the country to w h i c h the
largest
proportion of questionnaires was sent.
Secondly
it
wo u l d s e e m
incorrect in trying to establish the
m o s t
reliable
procedure
for chlorophyll
a
analysis to d r a w attention to a piece of
a ppa ra t us
or procedure
m o s t c o m m o n l y used w h e n it is the p u r p o s e of this
investigation
to obtain an
objective appraisal of only
w h a t
is best.
Ill A
suggested
procedure for the establishment of a
standard method for the determination of chlorophyll a
in
sea-water
'
A
A P P A R A T U S
1 Spectrophotometers and
colorimeters
Since m a x i m u m
sensitivity of the determination requires
m a x i m u m extinction
of
light per unit weight of c o m p o u n d to be analysed, the use of
optical
e q u i p m e n t
with b r o a d w a v e - b a n d widths , wide
slit
widths or wave- length settings
which
a r e difficult
to adjust, should be discouraged.
S o m e
types of spectrophotometers
m e e t these
requirements
to a greater or
lesser degree,
but colorimeters are of
limited
use in waters of low
p i g m e n t
concentration
because
the
b r o a d b a n d - p a ss
o f the filter eads to red uc ed
sensitivity.
It is im p or tan t that the wave- length
setting of s pe c tro pho to m e te rs be routinely
c hec ked
(see suggestions of
N A S C O
report).
2
In
order
to p e r m i t intercomparison of different spectrophotometers,
1 T h e following
discussion is
ke ye d
to the information reported in A p p e n d i x I.
2
Excerpts from the
N A S C O
report and the
S C O R - U n e s co
intercalibration test are given at the
e n d
of
this
presen
tation.
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A survey of
m e t h o d s
th e optical
density at a
given wave- length should
be
standardized
in
te r m s
of
th e resolution of the instrument and the slit w id th th r o u gh w h ich th e
light
passes.
2 Light
path
of
cuvettes
T h e sp e ctr op h otom e te r e m p l o y e d sh ou ld
be
capable
of
a c c o m m o d a t in g
several
different
sizes of cuvettes.
W h e n
p i g m e n t values are k n o w n to vary over a w id e
ra ng e ,
light
p ath
lengths of 1
c m a n d 10 c m , a n d possibly a n intermediate length,
are required
for obtaining
optical
density
readings
in the
m o st accurate
portion
of the
scale.
3
Type of filter for removing plankton from
sea-water
It
m a y b e seen
in
A p p e n d ix
I
that
at
present there
is a
tenfold r a n g e
in
the pore
size of
filters e m p l o y e d
for
r e m o v i n g p l a n k to n
f r o m
sea-water.
In addition, the
material of
w h i c h
the filters are
c o m p o s e d
(not stated in
every
case)
m a y
be
variably soluble
in the
extraction solvent a n d ,
in
s o m e cases, m a y h a v e
a dele
terious
effect
on th e
light
transmission of the solvent. T h e typ e
of
filter e m p l o y e d
for r e m o v i n g p h y t o p l a n k to n f r o m sea-water should be standardized therefore,
a n d
in
addition m a d e readily available titTäTI o c e a n o g r a p h e r s
(cf. r e c o m m e n d a ti o n
of the S C O R - U n e s c o intercalibration test a n d N A S C O report) .
1
4
A pproxima te
suction
pressure
T h e
use of
high
suction
pressure
has
b e e n f o u n d
to
d a m a g e p h y t o p l a n k t o n
during
the
course
offiltration.A
m a x i m u m
suction
pressure
to be
applied
to
p l a n k t o n filters should
be
determ ined experimentally, taking into a c c o u n t
the
r e c o m m e n d a t i o n s
of the S C O R - U n e s c o intercalibration tests a n d the N A S C O
report.
5
Bonification a n d
grinding
apparatus
T h e r e is
s o m e evid en ce (N e lson , 1 9 6 0 ; L a e ssae an d H a n s en , 1 9 6 1 ) that
the use
o f sonification ap p ar atu s is necessary for the com p l e te extraction of p i g m e n t
f r o m s o m e
species
of
p h y t o p l a n k t o n .
In addition it is
n ote d
in A p p e n d ix I that
s o m e
p e r son s
e m p l o y
grinding
ap p ar atu s
w h i c h ,
if
f o u n d
as effective as sonifi
cation,
should
be
given prior
r e c o m m e n d a t i o n on the
basis
of its
lower cost.
A t h o r o u g h
testing of the effect of
sonification a n d grinding a p p a r a t u s
on a
series
of
natural p h y t op la n k to n b l o o m s a n d
a
standard m i n i m u m treatment
(for
sonification
ap p ar atu s ,
in
te r m s
of period of
treatm ent, frequency a n d energy
o f
sonifier)
should be determined
if
f o u n d
necessary.
B R E A G E N T S
1
Solvent with which cells a re
extracted
S o m e evidence
exists
(L ae ssoe an d H a n s e n , 1 9 6 1 ;
see
also
N A S C O report)
that
m e t h a n o l
is a
better
solvent for the extraction of m a r i n e p h y t o p l a n k t o n t h a n
1
Excerpts
from the
N A S C O report
and the S C O R - U n es co
intercalibration test
are
given
at the end
of
this
presen
tation.
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Determination of ph otosynthetic pigments
in
sea-water
9 0 per cent acetone.
A
c o m p a r i so n
of
these two
solvents
should be
m a d e
on
a
series
of
natural p h y t o p l a n k t o n
b l o o m s
and the best solvent r e c o m m e n d e d
for
routine use.
2 Addition of basic material during extraction
F o r preventing the formation of
p h e o - p ig m e n t s
M g C O s is
often
a d d e d during
extraction. This should be c o m p a r e d for effectiveness with dimethylaniline w h i c h
h as b e e n
reported to be a
better
additive for this p u r p o s e (Vallentyne, 1955;
Patterson and P a r s o n s , 1963) .
C P R O C E D U R E
F o r
discussions
of v o l u m e of sea-water filtered (C.l) and chlorophyll c o n c e n
trations
encountered ( C . 2 ) , see discussion on
precision
of chlorophyll a deter
mi n a t i o n s (E.4)
bel o w .
3
Desiccation
of
filters
prior to
extraction
T h e
n e e d for
desiccation
prior to
extraction
should be
d e m o n str ated
experi
mentally. A standard m i n i m u m treatment should be f o u n d
if
desiccation
is
s h o w n
to be necessary.
4
Steam
treatment of filters
S t e a m
treatment of
s a m p l es
has
b e e n
e m p l o y e d
by
a
n u m b e r of scientists, presu
m a b l y
to
prevent formation of chlorophyllide
f r om
chlorophyll by the action
of
chlorophyllase. Since chlorophyllide a has
been
reported
to
h a v e the s a m e spec
t r u m and extinction
coefficients
as chlorophyll a (Holt a n d J acob s ,
1954) ,
the use
of ste am w o u l d a p p e a r
to
be an unnecessary step. The effect
if
any should
be
d e m on str ate d experimentally on natural populations a n d on Skeletonema costatum
w h i c h
has been reported
to
h a v e
a
very high chlorophyllase
activity
(Patterson
a n d
P a r s o n s ,
1963;
Jeffrey,
1963) .
5 Storage of
filtered
sample
T o g e t h e r
with C . 3 , covering
the
desiccation
of s a m p l es , the
preservation
of
p l a n k t o n s a m pl e s for
different periods
of
time should be tested experimentally.
A
m a x i m u m
storage period of three m o n t h s w o u l d s e e m , if experimentally
possible,
sufficient
for scientists on ships
w h i c h
do not h a v e
facilities
for carrying
out all parts of the
procedure
on b oar d .
6
Type
of
container
used to
carry
out
extraction
T h e
facility of
extraction, centrifugation and
v o l u m e
adjustment
in
glass-stop
pered
graduated
centrifuge
tubes should be
c o m p a r e d
with other apparatus and
a standard extraction vessel r e c o m m e n d e d . This consideration probably
has
little effect
on the
precision
and accuracy of the m e t h o d but for
laboratories
starting
p i g m e n t w o r k
it is
useful
to
k n o w the best pieces of a ppa ra tus
to
order.
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A survey
o f methods
7
Length of extraction time
In c o m b i n a t io n with items C . 9 a n d A . 5, covering the use
of
apparatus e m p l o y e d
to rupture
cells,
the m i n i m u m period of time required for an extraction, and the
benefit
if
any
of
hot extractions, should be
f o u n d
experimentally with the use
of natural populations. It is possible
that
a long
extraction
period without the
use
of a ppa ra tus
to
rupture
cells
m a y be f o u n d equivalent
to a
very short extrac
tion with such apparatus . Equivalent extraction procedures should be r e c o m m e n
ded
as alternative
p rocedures .
8
Volume for extraction solvent
Discussed
u n d e r
E . 4 , Precision.
9 Methods employed to rupture cells
Discussed
u n d e r
C . 7
and A . 5 .
10 Removal of extracted material
T h e
use
of
filters
c o m p a r e d with
centrifugation
for the
r em o v a l
of
extracted
material should be
e x a m i n e d
with
attention
being paid to the
facility
of operation
a n d the
efficiency
of r em o v a l
of
extracted material.
11
B l a n k
employed
of
0
optical
density
T h e choice of a suitable 'blank' for 0 optical density should be
m a d e
experiment
ally
bet w een
the use of the extraction
solvent
a n d the use of the
solvent
plus filter
material and any additive
to
prevent peophytin formation.
12
W a ve - l e ng t h s
at
which
measu rements are made
A s K r e y observed in 1958 (loc. cit.), the determination of chlorophyll a by tri
c hro m a t i c spectrophotometry
is
only slightly affected by chlorophylls
b
and
c.
If
all
three chlorophylls are present
in
equal
a m o u n t s
the
m a x i m u m
error
in
the
estimation of chlorophyll
a
by
a
single 66S my. reading
in
90 per cent acetone
is
a b o u t
10 per cent. Since
m o s t
of this error
is
contributed by chlorophyll b which
is generally absent from oceanic sea-water sam p l e s ,
the
actual error in m a k i n g
a
chlorophyll
a
estimation uncorrected
for
other chlorophylls
is
not m o r e than
a b o u t
1
per cent. For chlorophyll a determinations alone, therefore,
a
single
optical
density reading m i g h t
be
r e c o m m e n d ed
for the m e a s u r e m e n t of the
p i g m e n t .
A correction for turbidity should be introduced by m a k i n g a m e a sur e
m e n t
at
750 mji and the establishment
of a
standard p r oce d u r e
for a
750
my.
correction
should
be
considered along the
Un e s
r e c o m m e n d e d
by
the
N A S C O
report and the S C O R - U n e s c o intercalibration
test.
13
Extraction performed
Be cau se of the limited time and space
available
o n
s o m e
ships for the completion
of all parts of the
procedure
on b o a r d it is necessary
that
the
final procedure
be
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D e t e r m i n a t i o n o f photosynthetic p i g m e n t s in s e a - w a te r
written to give a n
indication
o f wh e re it is
possible
to break o ff a n d co m p let e
th e
analysis
on shore. It is r e c o m m e n d e d that this should be considered un d e r
C . 5
a n d
C . 3 .
D
S T A N D A R D I Z A T I O N
1 Extinction coefficient employed
fo r
chlorophyll a
T h e choice of a
suitable
extinction coefficient for chlorophyll
a
should be m a d e
f r o m th e large n u m b e r o f values quoted in the literature. F o r this purpose it is
r e c o m m e n d e d that
the value quoted by S m i t h and Benitez (1955) which agrees
with the value of Zscheile a n d C o m a r (1941) of 10 2 1/g c m in
ethyl ether
at
662
m
(j. should be given
p r i m a r y
consideration.
This value is suggested by
S m i t h
a n d Benitez (1955) for use as a standard
since
in a n extractive spectrophotometric
procedure , chlorophyll
a
is not dried in the extracted
state.
Ch lo ro p h y l l
a
w h i c h
has
been dried in the extracted state w a s fo u n d b y Zscheile a n d C o m a r (1941) to
give a lower specific absorption coefficient than undried chlorophyll . T h e specific
absorption coefficient in 90 per cent acetone corresponding to the value quoted
a b o v e
in
ethyl ether
h a s b e e n f o u n d b y
V e r n o n
(1960) to b e 91 1/g c m at
664
m [ i .
1
Fol lowing the choice o f a n extinction coefficient for chlorophyll
a
it should
n o t b e
r e c o m m e n d e d
that
a
c o m m e r c i a l
preparation of
chlorophyll
a
b e us ed
as a p r i m ar y standard. S o m e
c o m m e r c i a l
preparation of
chlorophyll
a ,
or of a
m o r e stable derivative
s u c h a s p h e o p h y t i n , m i g h t b e
r e c o m m e n d e d ,
h o w e v e r ,
as a secondary standard with which to c o m p a r e
optical
densities as described
a b o v e ( A . l ) .
2 Extinction
coefficients
employed
fo r
other
pigments
estimated
Discussed
un d e r
F . l .
E C A L C U L A T I O N S O F R E S U L T S
1
Turbidity
correction
Discussed
u n d e r C . 1 2 .
2 Correction made
fo r
other chlorophylls
a t wave-length f or
chlorophyll a
Discussed un d e r C . 1 2 .
3
Correction
fo r
degradation products
of
chlorophyll
a
It w o u l d be very
useful
to h a v e
s o m e
m e a s u r e of the
a m o u n t
of degradation
p ro d u cts o f chlorophyll
a
present in
m a r i n e
s a m p l es
since
if these are appreciable
1 If a decision is
m a d e
t o e m p l o y m e t h a n o l as a n
extraction
solvent th e specific absorption coefficient o f chloro
phyll
a i n m e th a n o l will have to be determined in a
m a n n e r
similar to that e m p l o y e d b y V e m o n (1960) for the
determination of the specific
absorption
coefficient of chlorophyll a in 90 per cent acetone.
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• \
survey
of
m e t h o d s
they will cause an erroneous over-estimation of chlorophyll a. At present there
a ppea rs
to be no reliable quantitative m e t h o d for su ch an estimation to be incor
po ra te d
in a
standard
pro c ed ure for
chlorophyll a analysis
of
sea-water samples .
T h e
introduction
of
s o m e
technique
at a later
date
w o u l d s e e m
advisable.
4
Precision of the chlorophyll
a
determination
T h e precision of chlorophyll a determinations is considered here in conjunction
with
the a m o u n t of sea-water
filtered
(C.l), the ra ng e of chlorophyll
a
values
encountered ( C . 2 ) , the v o l u m e of the extraction solvent
( C . 8 )
and the light path
length of cuvettes ( A . 2 ) .
T h e
precisions of chlorophyll a determinations quoted in A p p e n d i x I (E.4)
have been
taken
as
representative
of a
n u m b e r
of
values
q u ote d
by scientists,
often
in the a b s e n c e of
an explanation
of w h a t the
precision q u ote d
actually
m e a n s in statistical
terms.
A m o r e
detailed
description of precision in relation
to
v o l u m e
of sea-water filtered, ight path of cuvettes and
v o l u m e
of extraction
solvent
m a y
be considered as follows.
T h e
precision
for
chlorophyll a determination
at the 5 [/.g level
reported
by
Strickland and P a r s o n s (1960) is approximate ly ± 5 per cent.
1
E m p l o y i n g the
s a m e extinction coefficient for chlorophyll
a
that w a s used in those
calculations,
the
optical
density reading for this
a m o u n t
of
p i g m e n t
in 10 ml of extract and
using a 10 c m cuvette is a b o u t 0.33. If it m a y be
a s s u m e d
that optical density
readings
d o w n
to
a b o u t
0.1 can
be
m e a s u red
without introducing
a
decrease
in
the precision to
m o r e
than a b o u t ± 10 per cent, then the lower limit of
p i gm e n t
detection at this order of precision, empl o y ing the extinction coefficient suggested
in D.l a b o v e , is a b o u t 1
m g / m
3
if
1
litre of sea-water is filtered for extraction of
the residue with 10 ml of solvent and for an extinction
read
in a 10 c m cuvette.
T h e lower limit of
p i g m e n t detection
at the order of
precision
stated
a b o v e
can
b e decreased to 0.1 m g / m
3
if 10 litres of sea-water are filtered. It is probable , there
fore, that this value represents the lower limit of chlorophyll
a
values
which
s h o uld be quoted by persons using extractive s pe c tro pho to m e try in order that
all results
m a y
be
considered
to be
c o m p a r a b l e,
that
is,
obtained with
the
s a m e
order of
precision. Modifications such as reducing
the
extraction solvent
to 5
ml
(but maintaining a 10 c m light p a t h ) or filtering 20 litres of sea-water will almost
certainly introduce
difficulties
a n d
unnecessary
delays in p ro c edure for an increase
in the limit of detection
b y
a factor of only
t w o .
It
m i g h t
be considered advisable,
therefore, that chlorophyll a values of less t h a n 0.1 m g / m
3
should be reported
a s <
0.1
m g / m
3
and
not as s o m e
actual value w h i c h w o u l d not be c o m p a r a b l e
with p i g m e n t
values determined
a b o v e
the limit of detection quoted here. In the
table
b e low
the l ow e r limit of chlorophyll
a
detection,
a s s u m i n g
a b o u t ± 10 per
cent precision,
is
s h o w n
for
various
c o m b i n a t i o n s
of
cell
lengths and
v o l u m e s
o f sea-water
filtered
a n d
a s s u m i n g
10 m l of solvent are
e m p l o y e d
for the extrac
tion. T h e
table e m p h a s i z es the
necessity
for the use of 10
c m
light paths for the
determination of p i g m e n ts in the range 0.1 to 1.0 m g / m
3
.
1 See page 5 of reference quoted for an explanation of this value.
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A
survey of m ethod s
matic spectrophotometry on oceanic p igm e n t sam p l e s . W h e n one considers
that
the optical density at 630
m\x
contributed by 1 ¡¿g of chlorophyll
c
in 10 ml of
solvent using a 10 c m cuvette is only a b o u t 0 . 0 2 , and
that
the value of 1 ¡xg is
pro ba bl y
m o r e
tha n
will
no rm a l l y
be encountered in
ocean
areas, it is not sur
prising
that
s o m e extraordinary ratios of chlorophyll c : a h a v e been reported
for o c e a n areas (see
accu m u l ate d
values by
H u m p h r ey ,
1 9 6 1 , for ex a mpl e ) w hic h
h a v e not been confirmed with determination on p h y t o p la n k t o n cultures. In the
case of estimations of plant carotenoids, the
difficulty
recognized by Ri c ha rds
with
T h o m p s o n
(1952) of having to
e m p l o y
specific p i g m e n t
unit
is complicated
further by the reported use of a different specific p i g m e n t
unit ( A p p e n d i x
I,
D . 2 )
tha n that
originally defined by R ic ha rds with
T h o m p s o n
(1952).
I n view of these c o m m e n t s it
w ou ld s e e m
that
the
best
w a y to obtain m a x i
m u m benefit
from
the extracted
p igm e n ts ,
other
t h a n
for the estimation of chlo
rophyll
a,
is to read extinctions at certain other wa ve-lengths but not to interpret
these readings in term s of absolute
a m o u n t s
o f
p i g m e n t .
T h u s it m i g h t b e suggested
that
in addition to a reading at 750 m p i and 665 m \ > . for the estimation of
chloro
phyll a, additional optical densities should be read at 645, 630, 510 and 480 m\i.
Further readings
that
m a y eventually
p r o v e
useful w o u l d be at 505 and 430 m ( x .
Ratios of optical densities at these wave-lengths
m a y p r o v e
m o r e reliable than
attempting to
determine
the pigments involved in absolute a m o u n t s . M e a s u r e m e n t
o f
the entire
spectrum
of p i g m e n t extracts w o u l d present the
best solution
to this
p r o b l e m but
this
is undo ubte dl y too tedious for routine analysis except
w h e n
a
specific
study is being
m a d e .
2
Use of a fluorometric echnique for
routine
determinations
F o r s o m e oceanic areas (e.g. Sargasso Sea) the
limit
of chlorophyll a
detection
o f 0 .1 m g / m
3
(discussed
a b o v e ( E . 4 ) ) m a y not be low
e n o u g h
to s h o w seasonal
a n d spatial differences in chlorophyll a concentrations. In such areas it m a y be
advisable to
e m p l o y
a fluorometric technique, since the limit of
detection
for
fluorometric m e a s u r e m e n t s of chlorophyll a is at
least
ten times lower
t h a n
for
spectrophotometric
m e a s u r e m e n t s .
Fluorometric estimations include all chloro
phylls, h o w e v e r , and thus the results are not strictly c o m p a r a b l e to a spectro
p h o t o met r i c
technique for chlorophyll a alone.
O n
the other h a n d it has been
fou n d possible
to give s o m e m e as u r e of the proportion of chlorophyll
degrada
tion
products by fluorimetry (Yentsch and M e n z e l, 1963) w h i c h has
b e e n m e n
tioned here (E.3) as one desideratum for the spectrophotometric determination
o f chlorophyll
a.
I n
reaching a conclusion on the
desirability
of using a
fluorometric
technique
for chlorophyll determinations on a routine
basis
it
m a y
be advisable to suggest
that
a
sufficient
n u m b e r
of
spectrophotometric
m e a s u r e m e n t s
should
b e
perf o rmed
to characterize an area of low chlorophyll content (i.e. < 0.1
m g / m
3
)
and that
a m o r e detailed description could then be presented in te r m s of fluorimetric
determinations. A suitable m e th o d for the fluorimetric determination of chloro
phyll in sea-water has been reported by Kalle (1951) and another by Y e nts c h
a n d
M e n z e l
(1963), w hic h is a modification of Kalle's technique.
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Determination of photosynthetic pigments in sea-water
EXCERPTS
A N D R E F E R E N C E S
Excerpts
from
t h e
NASCO report
entitled
Recommended Procedure
for the
Measurement
ofPhyto-
plankton Pigments prepared by the N A S I N R C Committee on
Oceanography
Working Group on
Standardization
a n d
Inter
calibrât
i o n o f
Biological
Measurements
a n d
Sampling
Methods ,
28
March
1 9 6 3
With
reference
to :
Section
A.l 'It is
recommended that
the spectrophotometer be calibrated frequently using
narrow
band-pass filters or D i d y m i u m glass (or
equivalent).'
Section
A.3 'Th e water samples collected for phytoplankton pigment
analysis
should be
filtered
through cellulose-type membrane filters e.g. Millipore R Type
H A
or
P H ,
or
equivalent) or
possibly fine glass-fiber
filters e.g.
W h a t m a n G F / C ,
Gelman
glass filters,
or equivalent).'
Section
A.4
T h e pressure
reduction
should not exceed 50c m H g .
Section
B.I 'Methanol and diethylether extract phytoplankton pigments
better
than acetone.'
Section
C.12
'Following
extraction,
acetone solutions should be
centrifuged
so that the optical
density at
7 5 0 m j j.
is less than 0.005/cm of path length of
light, after
the blank has been
subtracted; the optical density at 7 5 0 m\i must be kept below 0.01.'
Excerpts from SCOR-Unesco
intercalibration
test entitled Circular Memorandum to National
Committees,
Indian Ocean
Investigations ,
9 January 1962
With
reference to :
Section
A.3 Filters should be soluble in 90 per
cent
acetone, should have a pore size of no
more than 0.8 n, and should not be subjected to high vacuum during
filtration.
Section
A.4 'The reduction in pressure should be about
V -
t 0
1
h of an atmosphere.'
Section
C.12 'If the
optical
density
at 750 mi¿ is
greater
than
0.005/cm
path,
recentrifuge,
refilter, or dilute to reduce this reading.'
H O L T , A . S . ; J A C O B S , E . E . 1 9 5 4 . Spectroscopy of plant pigments. I Ethyl chlorophyllides A and B
a n d their
pheophorbides.
Amer.
J. Bot., 41.
H U M P H R E Y , G . F. 1961. Phytoplankton pigments in the Pacific Ocean. Preprint, Symposium
o n
algal productivity
in the Pacific, 10th Pac. Sei. Congr., p. 16.
J E F F R E Y , S . W .
1963 (results to be published).
K A L L E ,
K . 1 9 5 1 . Meereskundlich-chemische
Untersuchungen mit Hilfe des Pulfrich-Photometers
v o n
Zeiss VII. Die
Mikrobestimmung
des Chlorophylls und der Eigenfluoreszens des
Meerwassers. Deutsch. Hydrogr. Zeitschr., 4.
K R E Y ,
J. 1 9 3 9 . Die Bestimmung des Chlorophylls in
Meerwasser-Schöpfproben, J. du
Cons., 14.
. 1 9 5 8 .
Chemical methods of
estimating
standing crop of phytoplankton.
Rapp.
et Proc-
Verb.
Cons, internat.
Explor. de la Mer,
144.
L A E S S O E , A . ; H A N S E N ,
V a g n
K r . 1 9 6 1 . Ultrasonic
and extraction of chlorophyll a from phyto
plankton. Plankton Committee Report N o . 143. Cons, internat. Explor. de la Mer.
N E L S O N ,
D .
J.
1 9 6 0 .
Improved chlorophyll extraction
method.
Science, 132.
P A T T E R S O N ,
J.;
P A R S O N S , T . R .
1963. Distribution of chlorophyll a and degradation products
in various marine samples.
Limnol. Oceanogr.
(in
press).
R I C H A R D S ,
F.
A . W I T H T H O M P S O N , T . G .
1952. The
estimation
and characterization of
plankton
populations
by
pigment analysis.
II. A
spectrophotometric
method for the estimation
o f
plankton pigments. / . Mar. Res., 11.
S M I T H , J. H . C ; B É N I T E , A . 1955. Chlorophylls. Analysis in plant
materials.
Modern methods
o f
plant
analysis, vol. IV, p.
143-96.
Berlin, Springer-Verlag.
S T R I C K L A N D , J. D . H . 1 9 6 0 .
Measuring
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