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Translation Series No. 3856

Methods for quantitative determination of chlorophyll

•

by Y. Saijo

Original title: Kurorofiru no sokuteiho

From: Rikusuigaku Zasshi 36(3): 103-109, 1975

Translated by the Translation Bureau(JWC/PS) Multilingual Services Division

Department of the Secretary of State of Canada

Department of the Environment Fisheries and Marine Service

Halifax Laboratory Halifax, N.S.

1976

26 Pages typescript

ene 2t4.

tkerelie CANADA

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DIVISION MULTILINGUES

AUTHOR - AUTEUR

Yatsuka SAIJO TITLE IN ENGLISH - TITRE ANGLAIS

METHODS FOR QUANTITATIVE DETERMINATION OF CHLOROPHYLL

TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGERE (TRANSCRIRE EN CARACTÈRES ROMAINS)

Kurorofiru no sokuteiho

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Rikusuigaku Zasshi

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Japanese Journal of Limnology, 3 , 103 - 109, 1975

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1101531 Japanese JWO:'^ S.

Japanese Journal of Lirrnology, 36, 103-109, 1975•

Series on exnerimental methods in 1.imnologV, No. I.

Methods for quantitative determination of chloroph,yll.

Yatsuka SAIJO.

p103

1. Introduction.

M-

More than 30 years have elapsed since the first use

of measurements of the amount of chlorophyll in water for the

determination of the quantity of microalgae, or, better, of

phytoplankton, in both inland and ocean waters. The earlier

methods of measurement have been discussed in detail by Krey

(1958) but at that time they could only be called semi-

quantitative. Since then, tremendous advances in the techniques

of measurement of chlorophyll have been introduced through the

develop:^.ent of membrane filters and glass fibre filters for

filtration and concentration, and through advances in measurin;

,F^ 5_75T (6176)0

2

eauipment for analysis by colorimetry or fluorescence. In

addition, r^easurements of chlorophyll have now become connected

to the problems of the quantitative evaluation of water-borne

resources and of eutrophication, and are being made by many

people.

In contrast to the measurement of nutrient salts in

water, there are no standard appropriate rea^ents for the

measurement of chlorophyll. The methods differ in convenience

and there are many points of procedure where error is possible.

The insufficiency of fundamental knowledge of plant pigments

sometimes leads to incorrect handling of the measured values,

and we therefore wish to discuss in some detail a summary of

the various methods which have recently been used, including

their uncertainties.

1. The chloroprVll in microalgae.

As is well known, all plants contain chlorophyll a,

but chlorophyll b and c also occur in many plants. Among the

microalgae only the blue--areen algae contain only

chlorophyll a.The green algae contain chlorophyll b, and the

diatoms, the dinofla-cellates, the yellow and brown flagellates

contain chlorophyll c. In addition, in lakes with some

eutrophication and in those with restricted circulation,

photosynthetic sulphur bacteria occur at the upper edge of the

anoxic layer and contain bacteriochlorophyll, but they will

not be included here. In the open ocean, green al7ae are in

3

general rare, and chlorophyll a and c are mostly measured,

but in inland waters green algae are common and chlorophyll a,

b, and c must all be considered. However the detailed analysis

of pigments by chromatography is laborious and requires

considerable skill and is not a suitable technique for routine

use. The Richards and.Thompson method of measuring chlorophyll

by means of light absorbtion was developed to solve the problem.

The chlorophyll is measured because of its relation to

photosynthetic production. Even -axxigh light is also absorbed

by chlorophyll b and c and by carotenoid, in fact the light is

transferred into photosynthesis by chlorophyll a, so that our

objective is satisfactorily attained if chlorophyll a is

correctly measured. For this reason, particular attention will

be given here to the measurement of chlorophyll a. As the

handbook of water analysis by Strickland and Parsons (1968) is

extremely detailed we shall make frequent reference to it in

the present paper for many details concerned with measurements

in both ocean and inland water.

2. Chlorophyll degradation products.

One important problem connected with the measurement

of chlorophyll in natural waters is that among the chlorophyll

pigments contained in a water sample there is also a greater

or less amount of its degradation products. There are two ways

in which chlorophyll is degraded. That which concerns the

magnesium leads to phaeophytin a, that which concerns the

phytol leads to chlorophyllide a. Both are then transformed

to phaeophorbides. It is not easy to make separate measurements

of these individually, but discrimination in measurement

between chlorophyll a and phaeophytin a (or, strictly

speaking, phaeopigments containing phaeophorbides) is fairly

easy. However it is difficult to make separate measurements

of chlorophyll and chlorophyllide because they have the same

light absorbtion.

p104 2. The measurement of chlorophyll a, b, and c

by light absorbtion.

This method, which is known as the Richards and

Thompson (1952) method or the "trichrometric" method, has been

improved by Parsons and Strickland (1963) and by the SCOR/UNESCO

(1966) working group on photosynthetic pigments. We will here

mostly follow Parsons and Strickland (1961)* and describe the

methods which we use.

1. Outline of the method.

The water sample is filtered, the deposit on the

filter paper is extracted with acetone, the extracted liquid

is separated by centrifuging, the light absorbence of the

supernatant liquid is measured with the use of a light absorbtion

meter, and the quantities of chlorophyll a, b, and c are

• calculated from the measurements.

* Sic. I cannot find this paper,.but the details given suggest a misprint for Parsons and Strickland (1963). Translator.

5

2. Equipment.

(1) Filter and suction nurp.

A 47 mm filter is satisfactory, and, for field use,

a plastic filter which is difficult to break is advantageous. At

aspirator is used for suction pumping, but there is little

trouble with the recently introduced diaphragm type combination suctio:

and exhaust pump such as the Iwaki type AP-200.

(2) Spectrophotometer.

Hip;h sensitivity equipment is required. A small

volume cuvette with a light path of at least 5 cm and if

possible 10 cm is necessary.

(3) Electric centrifug-e.

A centrifuge which takes a number of 10 to 15 m%-

centrifuge tubes is satisfactory. A rotation speed of 3000 rpm

is sufficient.

(4) Grinder.

Small, diameter about 8 cm.

4. Rea.7ents, etc.

(1) 90I ( v/v) acetone solution.

( 2) Magnesium carbonate suspension.

Suspend 0.5 g of pure MgCO3 in 100 mt of pure water.

Shake well before each use.

6

(3) Filter paper.

Use 47 mm diameter glass fiber filtPrs such as WhatTnan GF/C or

Reeve Angel 984H ultrafilter.

4. Method.

(1) Filtration.

The water sample is suction filtered through a glass

fibre filter of 47 mm diameter. If 1 m.e, of the magnesium

carbonate suspension is added beforehand to the water sample,

and well shaken before filtering, the conversion of chlorophyll

to phaeophytin is prevented and it is also effective in

increasing the proportion of suspended matter collected. It

is considered satisfactory to determine the size of the water

sample filtered by dividing the transparency of the water at

the point of collection (in metres) by five and taking that

number of litres. For the same transparency, freshwater is

more difficult to filter than seawater, but the suction

pressure should not go below one-third of an atmosphere.

After the water has been sucked through the filter,

suction is continued until the filter is dry. When it is not

possible to submit it to chemical analysis immediately, it

should be cooled and preserved at -20 °C. It is said that

there will be no change for two to three weeks, but it is

safest to analyze as soon as possible. Furthermore, if dry

ice is used for freezing, it is to be feared that the carbon

dioxide will convert the chlorophyll to phaeophytin. If its

use is unavoidable, freeze the sample after putting it into a

perfectly tightly sealed container.

(2) Extraction.

The edges of the filter paper are cut off with scissors

and discarded. The filter paper is then cut into small pieces

and placed in the grinder. A small amount of 90% acetone is

added to make it damp and it is thoroughly ground. If this

operation is not complete the extraction will be insufficient.

The contents of the arinder are washed into a graduated glass

centrifuge tube, with 2 or 3 repetitions of small amounts of

90% acetone. It is advantageous to do this through a small

funnel. The mouth of the centrifuge tube is covered with

parafiim, and after incubating for about one hour in a cool

dark place a balance is added and it is centrifuged for

5 minutes at about 3000 rpm. Since the chlorophyll extract

is easily altered by light, care is taken to avoid a strong

light while these operations are in progress.

(3) Colorimetr .

Each cuvette to be used for colorimetry is previously

used to measure the light absorbence of the 90% acetone and

derive the appropriate correction. A known amount of the

centrifugally separated extract is taken in a pipette, placed

in the cuvette, and measured by the colorimeter. The quantity

of liquid extract remaining is measured in a small measuring

8

cylinder, and the ratio of the amount of liquid used for

colorimetry to the total amount of extract is found.

Readings of the light absorbence of the 90% acetone

extract are made at the following wavelengths:-

750, 665, 630, 480 mp...

The measurement at 750 y is made in order to check

the turbidity produced by substances other than plant pigments.

If the reading at the wavelength of 750 m),4, is greater than

0.005 for a 1 cm cuvette, the liquid is to be returned to the

settling tube, several drops of 100% acetone areto

be added, and the measurement is to be repeated after further

centrifuging.

(5) Calculation.

The measured value of the optical density to be used

for each wavelength is that obtained by subtracting the value

measured at 750 raft from that measured ateadh particular wave-

length. However, at 480 mityL only, three times the optical

density at 750 raja. is to be subtracted. Denoting these

corrected values by D, the following equations are to be used

to calculate the amounts of chlorophyll a, b, c, and of

plant carotenoid.

y' ,

9

Chlorophyll a = 11.6 D665 - 1.31 D645 - 0.14 D630

Chlorophyll b=-4•34 D665 + 20.7 D645 - 4.42 D630

Chlorophyll c=-4.64 D 665 - 16.3 D + 55 D630

Plant carotenoid

Plant carotenoid

= 4.0 D480 ......... (1)

= 10.0 D480 ......... (2)

P105

Equation (1) is to be used when the principal source

is green or blue-green algae, and equation (2) when the

principal source is diatans, yellcsw or dinoflagellates.

The values obtained from these equations are the

quantity of pigment, in micrograms, when the acetone extract

obtained by filtration of one litre of water sample is measured

with a light path of one centimetre. The individual amounts

of chlorophyll a, b, and c, and of plant carotenoids in mg/m3

are therefore given by multiplying the values obtained from

the above equations by a factor "f", where

Total amount of extract (millilitres)

f = x ...(6)#

Quantity of water filtered (litres) Cuvette length (cm)

* Sic, there are no equations 3, 4, or 5. Translator.

10

Notes.

(1) Membrane filters were formerly used, but an ultrasonic

process is then necessary for extraction and a centrifuge speed

of 15000 rpm is also necessary. This process completely

disrupts the cell membranes and also removes the turbidity

produced in the acetone extract by colloids from the membrane

filter. When glass fibre filters are used, the glass

powder acts as an abrasive, and there is no feer of colloids appearing so

that this method is simpler.

(2) The values obtained by this method are highly accurate

for chlorophyll a, but it is to be supposed that for chlorophyll b

and c and for carotenoid they are no more than rough estimates

for reference. The value for chlorophyll a itself contains

the values for its degraduation products. If chlorophyll a is

to be separated from phaeopigments, either Lorenzen's (1967)

improved method or the fluorescence method to be described on

page 14 is to be used. If, in the light absorbtion method,

exact values for chlorophyll c only are required, the method

of extraction to be used in conjmction and introduced by Parsons (1963)

may be folimed.

•

11

3. The separate determination of chlorophyll a

and phaeopigments by light absorbtion.

The light absorbtion method described above is that

which is most widely used as a standard method for the

determination of chlorophyll. However an important defect of

this method is that chlorophyll a cannot be separatalfrom its

degradation products. Moss (1967) and Lorenzen (1967) have

developed methods of determination of chlorophyll by light

absorbtion which are better in this respect. There is no great

essential difference between these methods, and we will present

an introduction to the method of Lorenzen, which is conbidered

to be the more practical.

1. Outline of the method.

In this method the light absorbence of the chlorophyll

pigments in the acetone extract is first measured. Acid is

then added to the extract, and the chlorophyll a is converted

to phaeophytin and the absorbence is again measured. The

quantity of chlorophyll a is obtained from the change (reduction)

in the absorbence.

2. Equipment.

The same as above.

3. Reagents, etc.

The same as above, together with 1N hydrochloric acid

(the concentrated acid is 12N).

12

4. Method.

The same as above, as far as the measurement of the

absorbence. After the absorbence of the extract has been

measured at wavelengths of 750 m^k and 665 m/t , two drops of

dilute iN HCl are added to each 5 ml of the extract. After.

leaving for three minutes, the absorbence is again measured

at the same two wavelengths. Special care must be taken in

washing out the cuvette after use, so that the acid will not

affect the next sample to be used.

5. Calculation.

The blank values are corrected by subtracting from the twice

measured values of 665 mp the corresponding values of 750 RYA and they are

represented as 665o and 665a. The quantities of chlorophyll a

and of phaeopigment are calculated from these values by means

of the following equations:-

Chlorophyll a- A x K x(6650 - 665a) ""'se (7)

Phaeopigment = A x K (R E665a) - 6650) .•••••• (8)

where

A = the chlorophyll a absorbtion coefficient, = 11.0.

K- a coefficient for calculation of the original

concentration of chlorophyll from the reduction

of absorbence after acid has been added.

It is 1.7/0.7 or 2.43

R the ratio of 665o/665a when there is no phaeopigment

(the maximum value) and is 1.7.

13

The values so obtained are the quantities, in

micrograms, contained in the acetone extract when a one-

centimetre cuvette is used and one litre of. 'water sample has

been filtered. To obtain the quantity of chlorophyll a and

of phaeopigment, they must be multiplied by a factor "f" where

Total amount of extract (millilitres) 1-

f - Quantity of water filtered (litres) Cuvette length (cm)

When the values of A, K, and R are inserted in

equations (7) and (8) we obtain

Chlorophyll a (mg/m3) = 26.7 (665 0 - 665a ) x (f)

Phaeopigment (mg/m3 ) = 26.7 (1.7 x 665a - 6650 ) x (f).

Note: Chlorophyllide a may be contained in the chlorophyll measured by this

method. In some phytoplankton, suCh as Skeletonema costatum,

Phaeodactylum tricornutum, and Dunaliella tertiolecta, in which

the chlorophyllide contains chlorophyllase, it is possible that

the chlorophyllide a may reach 10% to 20% of the chlorophyll a.

(Barrett and Jeffrey, 1964; Saijo and Kamiya, 1972). p106

p106

14

4. The determination of chlorophyll a and of phaeopigments

by means of fluorescence.

When chlorophyll or chlorophyll extract is illuminated

by ultraviolet light, a red fluorescence is produced. Since

the strength of the fluorescence is proportional to the

strength of the excitation, an increase of sensitivity of two

orders of magnitude over the light absorbtion method can be

obtained by the use of a strong source of light. In the

present discussion we mostly follow Yentsch and b:enzel (1963)

and Holm-Hansen (1965) and describe the methods which we use. '

1. Outline of the method.

Filtration and acetone extraction are the same as in

the light absorbtion method. The fluorescence of the extract

at wavelengths greater than 650 m/A is measured, acid is added

to the extract so that the chlorophyll is wholly converted to

phaeopigment, and the fluorescence is again measured. The

quantity of chlorophyll a is obtained from the difference

between the two measurements.

2. Eouipment.

(1) Filter and suction pump.

The same as in the light absorbtion method. However

a filter diameter of 24 mm may be used.

15

( 2) Fluorometer.

The filter type of fluorometer is satisfactory, but

the sensitivity of spectrophotofluorometers is low and they

are not suitable for routine use. The fluorometers which are

most easily used are those of the Turner Company in the United

States, which are widely used in Europe and America. In these

models there is a compensating circuit which makes the

measured values independent of reductions in the light output

of the exciting light source. In general the Turner type 110

is satisfactory, but in order to be able to make the continuous

recordings with a flow cell which will be described later, it

is better to buy the type 111. The cuvettes to be used with

fluorometers are normally made of quartz glass, but pyrex

glass is satisfactory for the measurement of chlorophyll, and

with the Turner fluorometers a low price round borosilicate

glass cuvette (12 x 75 mm, 3.5 mR ) can easily be used.

The source of ultraviolet light for the fluorometer

is normally a high pressure mercury lamp, its principal bright

lines being 365 m^n and 436 m^tn . As a source of excitation

for the measurement of chlorophyll, the wavelength 436 mrt,^ is

mostly used. The primary filter can be an interference filter

for 436 mIm , or a coloured glass filter with its maximum in

this region such as the Toshiba V-V44 or the Hoya B-43. With

the Turner fluorometer, the "blue lamp" light source may be

purchased with an adapter instead of a mercury lamp, and it may

be used in conjunction with the primary filter known as Corning 5-60,

16

A secondary filter is used on the side on which the

fluorescence from the sample is received. As can be understood

from Figure 1, a red filter such as ToshibaV-R65 or Hoya R-64

which passes only wavelengths longer than 650 mv. is used for

the measurement of chicrophyll. The filter naired asYecomMended for use with

the Turner equipment is the'Cornin 2.-LE4 calied secondary filter for chlorophyll

use, but there are filters by Shiboi and others with similar characteristics.

If the fluorometer is insufficiently sensitive, the

sensitivity can be increased by an order of magnitude by

substituting a photomultiplier such as the RCA R136 or the

Hamamatsu Television R446 which has high sensitivity in the

long wavelength region.

3. Reagents, etc.

These are the same as for the light absorbtion method,

with the additional need for 1N hydrochloric acid. When

fluorometers other than Turner models are used, a standardizing

solution of the sodium salt of fluorescein (1mgte ) is to be

prepared. This is made up as a 100 mg/Z solution, tightly

stoppered and stored in a dark place. It is appropriately

diluted for use. A glass fibre filter 24 mm in diameter is used.

}

17

30-i

20

JO

!/

-,- _

600 620 650 6-0

Fi--ure 1.

70'J 720mf1

Wave-length distribution of the fluorescence

excited by light of wavelength 436 m/(. in acetone extracts

of chlorophyll a, chlorophyll a + b and chlorophyll a + c.

The dotted lines show the wave-length distributions after

hydrochloric acid nas been added to the extracts and the

chlorophyll has been converted to phaeopigment.

18 p107

4. Method.

(1) Filtration.

This is the same as in the light absorbtion method,

but the quantity of water sample may be one tenth of that

for light absorbtion. For example one litre of water from

the regions of the Kuroshio current where there is least

phytoplankton is sufficient, and in lakes and marshes or in

inner bays where there is much phytoplankton 50 m/ is enough.

(2) Extraction.

The same as in the light absorbtion method.

(3) Measurement of fluorescence.

With ordinary fluorometers the sensitivity must be

maintained at a fixed level. The cuvette is filled with a

standard(1 mg/2 ) solution of the sodium salt of fluorescein,

and the fluorometer is adjusted to read 100% on the fluorescence

of this solution. Since the strength of the light from a

mercury lamp diminishes rapidly, checks and adjustments must

be made with the standard solution before and after the

measurement of a series of samples.

The standard solution is not required with Turner

fluorometers. An auxiliary dummy cuvette (a black bar) is

inserted, and when it is measured the dial is adjusted to zero.

The diaphragm stop used must be chosen to fit the concentration

in the sample, but since the ratios between the various stops

t

Phaeopigment (mg/M 3 )

19

differ somewhat from the figures given (lx, 3x, 10x, 30x) the

actual ratios for an individual meter must be previously found

by experiment.

In making a measurement, the total quantity of acetone

extract is first measured. A known quantity is then put into

the cuvette, and the strength Fo of the fluorescence is

measured. Next, 1N hydrochloric acid is added in the proportion

of two drops to 5 me and after it has been left for three

minutes the strength Fa of the fluorescence is again measured.

After the measurement, the cuvette is washed with special care

to ensure that the acid does not affect the next sample. Before

the measurement the cuvette is filled with the 90% acetone

solution and the fluorescence is measured in order to obtain

a blank value which must be subtracted from the other measured

values.

(5) Calculation.

Chlorophyll a (mg/M3 )

Where:-

Fo - Fa y

• — fph (R 1) V

RFa - Fo v • ---

fph (R - 1) V

R (the maximum value of the acid factor) = fch/fph

Fo is the original fluorescence reading

20

Fa is the fluorescence reading after the addition of acid

fch fDh are the characteristic fluorescences of chlorophyll ar

and of phaeôphytin, determined from known concentrations

of pure chlorophyll a and of phaeophytin a.

V is the quantity of water sampled (m^ )

V is the quantity of acetone extract (m ^).

(6) The determination of the meter correction factor.

Fluorescence readings are obtained from the acetone

extract of the sample with ordinary fluorometers which have

been set to 100% with the standard fluorescein solution, and

with Turner fluorometers which have been adjusted to zero with

the dummy cuvette and in which the characteristics of the stop

are known. The relation between these readings and the

chlorophyll a is in general to be obtained by the following

method.

300 m^ to 500 mi of a culture fluid are inoculated

with an easily cultured phytoplankton such as Skeletonema

which reaches its maximum growth in about five days. Acetone

extracts are made by filtering 100, 50, 25, 15, 10 and 5 mP

of this culture and the chlorophyll and phaeopigment are

determined by light absorbtion as described on page 11 . The

fluorescence readings Fo and Fa of the same extracts are

measured, and the fluoresecence readings of the samples are

corrected by comparison.

21

Note.

We wish to make some remarks about the acid factor.

When the fluorescence is measured, the ratio Fo/Fa between

the fluorescence Fo measured béfore acid is added and the

fluorescence Fa measured after acid is added is naturally

called the acid factor, and it reaches its maximum value when

there is no phaeopigment in the sample. However according

to Saijo and Nishizawa (1969) this acid factor varies with

the exciting wavelength as shown in Figure 2. In the vicinity

of 440 Irv,. it reaches a very large maximum of 10, but in other

regions it is less than 2. This is because the exciting

wavelengths which produce the maximum values of fluorescence

in chlorophyll a and in phaeophytin are, as shown in Figure 2,

slightly different.

When the Turner fluorometer is used with the blue

lamp and with Corning 5-60 as the primary filter and

Corning 2-64 as the secondary filter for the measurement of

an acetone extract in which chlorophyll a predominates and

there is essentially no phaeopigment, the acid factor obtained

is 2.0. When a very narrow band-width 436 mt interference

filter is used as the primary filter, the factor is about 5.

When natural samples contain blue-green algae, the acid

factor becomes excessive, and the quantity of phaeopigment

may appear to be negative.

22

o

.,--r---.---,-- -3;^ C..̂0 UA 49:mY

1YCvo len9t`, et excitir.g Iighl

12

10

EE

3^401. 440 490mIL\9ale tenglh of exiiGng Gght

Figure 2.

Above. The variations of the strength of the fluorescence

at the wavelength of 670 m^x of an acetone extract

of pure chlorophyll a, and of the extract after

conversion to phaeophytin a by the addition of acid,

as the wavelen.Eth of the exciting light is varied

from 360 mi/^, to 480 m^u

The dotted line shows the absorbtion characteristics

of the 436 m,^- interference filter.

Below. The ratio calculated from the above results of the

strength of fluorescence of chlorophyll a to that

of phaeophytin a (the acid factor) as a function

of each wavelength of the exciting light. The three

vertical lines show the positions and the relative

strengths of the main bright lines from a high

pressure mercury lamp.

2 3

The acid factor shows the abundance or scarcity of

chlorophyll degradation products in a phytoplankton community,

and can be taken as an indicator of the activity of the

community. For example, the chlorophyll pigments which have

passed through and have been discharged from the digestive

tracts of zooplankton are entirely phaeopigments. However the

amount of phaeopigment determined by the fluorescence method

is said to be frequently much greater than the amount accurately

measured by chromatography. Thus the phaeopigment values p108

obtained by this method risk being of narrow applicability

within restricted, semi-quantitative limits.

5. The direct determination of chlorophyll

pigments by fluorescence.

In the method developed by Lorenzen (1966) the

fluorescence of the unprocessed sample is measured by a

fluorometer in order to determine in vivo _ the quantity of

unchanged chlorophyll. This method is suited to the measurement

of a temporal or spatial sequence, but it has the defect that

the strength of the fluorescence of a unit quantity of

chlorophyll may be considerably affected by the phytoplankton

species (Strickland 1968), or in one species by the stage of

growth (Tunzi et al., 1974), and also by the presence in the

water of dissolved or suspended matter other than phylopimikton.

24

1. Outline of the method.

The sample is placed in a cuvette and its fluorescence

is measured without processing. Alternatively, the fluorometer

is equipped with a flow cell, and as the sample flows through,

changes of the quantity of chlorophyll are continuously

determined from the strength of the fluorescence.

2. Eguipment.

(1) Fluorometer.

The fluorescence of highly concentrated samples can

be directly measured with normal fluorometers, but the sensitivity

of the Turner fluorometers described on page 15 is high and they

are easy to use with attachments. With the Turner fluorometer

type 111, a 19 mm flow cell is used with a continuous flow-door

(#110-871) in order to make continuous measurement. The cuvette,

light source, and filters are the same as are described on page 16.

(2) Recorder.

A 10 mV full-scale recorder is used.

3. Method.

When the chlorophyll in individual water samples is

to be determined, they are simply placed in the cuvette and the

fluorescence is directly measured. When continuous measurement

is to be made a continuous flow-door is attached. Water is

taken up by a pump, and after.air bubbles have been removed

it is passed by gravity into the flow cell and the strength of

the fluorescence is continuously recorded.

11%

,

25

Before measurements are made, the flow cell is filled

with distilled water and the blank value is recorded on the

recorder chart. The ratio between the stops in the fluorometer

(lx, 3x, 10x, and 30x) must also be properly determined for

individual equipments. With continuous measurements errors

may be caused by fouling of the flow cell, and Lorenzen (1966)

recommends that the flow cell should be washed out daily with

a solution of potassium hydroxide in alcohol.

It is most essential that the readings obtained during

continuous recording should be correctly converted to

chlorophyll a values. For this purpose a definite quantity of

the sample flowing out of the flow cell should be collected and

filtered at prescribed intervals during the recording, or%eien

the values obtained show high or low concentrations. The collecting

times are marked on a recording shaet and the quantity of chlorophyll a

is accurately measured by the extraction method described on page 14, and

the results are used for the conversion into chlorophyll of the data

recœded by the direct method.

As already mentioned, the recordings of untreated

chlorophyll obtained by the direct method do not show more

than the relative variations, and the measurements also include

the chlorophyll degradation products. When the concentration

is high the strength of fluorescence is not proportional to

the concentration. Since the results of direct measurement are

not satisfactorily corrected by means of the extraction methods,

quantitative arguments based on them should be avoided.

à o

•

26

References.

BARRETT, J. and S. W. JEil, ::REY (196-1) Chloro-

phyllase and formation of an atypical chloro-

phyllide in marine algae. Pl. Physiol., 35; *.

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Hotm-IlassEs, 0., C. J. LORENZEN, R. w. OLMKS

and J. D. H. STRICKLAND (1965):Fluorometric

determination of chlorophyll. J. Cons. perin. Explor. Mer., 30: 3-15.

KEE; J. (1958):Chemical methods of estimating

standing crop of phytoplankton. Rapp. et

Proc.-Verb. Cons. Internat. Explor. de la

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LORENZEN, C. J. (1966): A method for the conti -

nuous measurement of in vivo chlorophyll

concentration. Deep-Sea Res., 13: 223-227.

LoasszEs, C. J. (1967):A note on the estimation

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PARSONS, T. R. (1963): A new method for the

microdetermination of chlorophyll c in sea-

water. J. Marine lies., 21: 161-171.

PARSONS, T. R. and J. D. IL Sralcm.Aso (1963):

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TUNZI, M. G., M. Y. Chu and R. C. BAIN, Jr.

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Yz.srscn, C. S. and D. W. MF.NZEL (1963) : A

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(Author: YPASUlIa SAW), Water Research Institute,

Nagoya University)