chlorophyll unesco

8/9/2019 Chlorophyll UNESCO

1/66


2/66

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 « «


3/66

Determinat ion

o f

photosynthetic

p ig m en ts

in

sea-water

Unesco


4/66

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


5/66

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


6/66

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 .


7/66

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


8/66

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


9/66

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


10/66

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

12


11/66

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.

13


12/66

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.

14


13/66

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


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


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


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


14/66

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


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


15/66

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


16/66

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

18


17/66

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


18/66

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.


19/66

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


20/66

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.

22


21/66

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.

23


22/66

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.

24


23/66

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


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

25


24/66

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


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.

26


25/66

• \

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.

27


26/66


27/66

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.

29


28/66

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

the production of marine phytoplankton. Bulletin No. 122

Fish. Res. Bd.

Canada, 1 7 2

p.

; P A R S O N S , T . R .

1960. A man

chlorophyll unesco

Documents