A Note on the Estimation of Chlorophyll a in Freshwater Algal CommunitiesAuthor(s): Brian MossSource: Limnology and Oceanography, Vol. 12, No. 2 (Apr., 1967), pp. 340-342Published by: American Society of Limnology and OceanographyStable URL: http://www.jstor.org/stable/2833052 .

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340 NOTES AND COMMENT

cystis aeruginosa having a lowest ratio of 0.59. A limited number of curves with lowest values spaced at intervals of 0.05 unit from 0.4-0.8 will be sufficiently com- prehensive to give percentage degradation over the range 0-90% to within + 5% for any extract.

EXAMPLES OF THE USE OF THE METHOD

Table 1 gives some values of 430: 410 ratios determined on 90% acetone extracts of mud and natural populations of algae in bodies of freshwater in England.

None of the extracts was completely un- degraded. Extracts of muds from Abbot's Pond, Somerset, and Shearwater Lake (Wiltshire) contain much pheo-pigment. Extracts of attached microalgae such as epipsammic diatoms also contain up to 40% degradation products, presumably de- rived from decaying but still attached cells. Extracts of epipelic algae harvested from sediment using the technique of Eaton and Moss (1966) show only small amounts of degradation. Since harvesting of these cells depends on their active movement into the tissue traps, theoretically only living cells are recovered. However, small amounts of mud containing decaying cells tend to ad- here to the tissues and probably these are responsible for the degradation products detected.

Extracts of net samples of actively grow- ing phytoplankton populations usually con- tain no pheo-pigments, but seston filtered onto glass fiber filters frequently contains degradation products. In the small pools studied, the water usually contained much fine suspended detritus which passed through the phytoplankton net (140-u

pore size) used. This detritus and also ultra- and nannoplankters may result in different 430: 410 ratios in extracts of total seston and of net samples from the same water. For example, net samples contain- ing only Pandorina morum had a lowest ratio of 0.69, whereas extracts of total seston had a corresponding ratio of 0.79.

BRIAN MOSS Department of Botany, University of Bristol, United Kingdom.

REFERENCES

BOGORAD, L. 1962. Chlorophylls, p. 385-408. In R. A. Lewin [ed.], The physiology and biochemistry of the algae. Academic, New York.

EATON, J. W., AND B. Moss. 1966. The estima- tion of numbers and pigment content in epipelic algal populations. Limnol. Oceanog., 11: 584-595.

FoGG, G. E. 1953. The metabolism of algae. Methuen, London. 149 p.

LIVINGSTON, R., R. PARISER, L. THOMPSON, AND A. WELLER. 1953. Absorption spectra of solutions of pheophytin a in methanol con- taining acid or base. J. Am. Chem. Soc., 75: 3025-3026.

PARSONS, T. R. 1961. On the pigment com- position of eleven species of marine phyto- plankters. J. Fisheries Res. Board Can., 18: 1017-1025.

RICHARDS, F. A. 1952. The estimation and characterization of plant populations by pig- ment analyses. I. The absorption spectra of some pigments occurring in diatoms, dino- flagellates and brown algae. J. Marine Res., 11: 147-155.

ROUND, F. E. 19,65. The epipsammon: a rela- tively unknown freshwater algal association. Brit. Phycol. Bull., 2: 456-462.

STRICKLAND, J. D. H., AND T. R. PARSONS. 1965. A manual of sea water analysis. Bull. Fish- eries Res. Board Can. 125, 2nd ed. 203 p.

A NOTE ON THE ESTIMATION OF CHLOROPHYLL a IN FRESHWATER ALGAL COMMUNITIES

The method of Richards with Thompson (1952) for the estimation of plankton pig- ments by absorption spectrophotometry has been widely used. It involves the measure- ment of absorbancy at three different wave- lengths for the estimation of chlorophylls

a, b, and c and is referred to as a trichro- matic method. Parsons and Strickland (1963) proposed modifications of the equa- tions used to calculate the amounts of pig- ments on the basis of redetermined specific absorption coefficients. The equations given

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NOTES AND COMMENT 341

in both papers depend for their validity on the absence of pheo-pigments in the extracts. According to Strickland and Par- sons (1965), pheophytins are generally absent from open ocean phytoplankton, but they may be present in considerable quan- tities in extracts of freshwater algal com- munities (Moss 1967). The method of Moss (1967) for the estimation of the fraction of chlorophylls degraded to pheophytins in plant pigment extracts gives only the ratio of chlorophylls to pheophytins, not absolute concentrations. This note describes a simple calculation for the estimation of the amounts of chlorophyll a and pheo- phytin a in 80 or 90% aqueous acetone extracts. I am indebted to Dr. F. E. Round for his encouragement and for criticizing the manuscript, and to Dr. P. Woodward of the Chemistry School, University of Bristol, for checking the equations. Support was provided by a grant from the British Na- tional Environmental Research Council.

The changes in absorption spectra that occur on conversion of chlorophylls to pheophytins decrease the accuracy of the trichromatic equations. These changes are relatively large at 645 and 630m/, the prin- cipal wavelengths used in the calculation of chlorophylls b and c, so the estimation of these chlorophylls in the presence of their respective pheophytins, by the trichromatic method, is particularly unreliable.

The presence of pheophytins b and c does not markedly change the absorption of an extract at 665m,u, the principal wavelength used in the estimation of chlorophyll a. The 665m/u absorption is similarly little af- fected by the presence of chlorophylls b and c, as has been recognized in the simpli- fied equations of Odum, McConnell, and Abbott (1958) and Talling and Driver (1961) that use only the absorption at 665m,u for the estimation of chlorophyll a. Therefore, in the estimation of chlorophyll a and pheophytin a using absorption only at 665mbu, the extract may be assumed to contain only these two components.

For a two-component system in which no chemical reaction is occurring, at a given wavelength A, with incident light intensity

Io and emergent light intensity I after pass- age through the absorbing system:

log Io - log I = Ck? + C'kct, (1)

(Kay 1964) where C and C' are the con- centrations of the two absorbers and kc and kc1, their respective specific absorption co- efficients, defined by the equation,

log (Io/I) = kcCd, (2)

where d = optical path length.

Also, log Io - log I = log (IO/I) = Absorbancy (AA). (3)

Let Ca be the concentration of chlorophyll a and P = concentration of pheophytin a, with kCa and kp the respective specific ab- sorption coefficients at wavelength x. From (1) and (3),

AA = Cakaa + Pkp. (4)

From the method of Moss (1967), the ratio Ca: P may be obtained. Let Ca/P = x. Then,

Ax = Caka a+-kp, and x

Ca=k?k. (5) xa=-kCa+ kp(5

Similarly,

AA = PxkCa +Pkp, and

P Ax (6) XkCa+ kp

Chlorophyll a and pheophytin a can then be estimated from a measurement at a single wavelength, provided their absorp- tion coefficients at that wavelength and the ratio Ca: P are known.

For 90% acetone, kCa has been deter- mined at 665m1. but kp has not. Vernon (1960) determined kCa at 665mbu and kp at 666mu, both in 80% acetone. Talling and Driver (1961) summarize evidence that k0a665 in 90% acetone probably differs by less than 3% from kCa665 in 80% acetone, and

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342 NOTES AND COMMENT

PHEOPHYTIN A I )0 so8I 60 4I0 20 0

O8 / 1 1 I 1 I I I I I 1 1 1

16-

14

12-

p ,lo-

a,ohyi a nm/ie,pote gis hoo

6-

4-

2

0' 0 20 40 60 80 100

% CHLOROPHYLL A

Fic. 1. The factors by which A665 (FA635) must be multiplied to give chlorophyll a and pheophytin a in mg/liter, plotted against chloro- phyll a and pheophytin a as percentages of total chlorophyll a + total pheophytin a in the extract. C-chlorophyll a. P-pheophytin a.

this is probably true for kp665. kp at 666mb also differs little from kp at 665mju (see Smith and Benitez 1955). Vernon's values (k0a665 = 90.8 liter g-1 cm-', and kp666 = 55.2 liter g-1 cm-') have been inserted in equa- tions (5) and (6), and the factors FA665 by which A665 must be multiplied to give the concentrations of pheophytin a and chlorophyll a in mg/liter using a 1-cm light path, have been calculated. The factors for single component systems of chlorophyll a and pheophytin a have been calculated using the general equation (2).

In Fig. 1, the calculated factors have been plotted against the appropriate per- centages of chlorophyll a and pheophytin a obtainable from the method of Moss (1967). This method gives the percentages of total chlorophylls and total pheophytins, the ratio of which, in a naturally degrading extract, may be assumed to be similar to the ratio 'of chlorophyll a to pheophytin a (x).

The factor for 100% chlorophyll a is cal- culated as 11.0 which is lower than that (11.9) of Talling and Driver (1961). Fu- ture standardization of specific absorption

coefficients will improve the precision of the FA665 values. Meaningful estimations of chlorophylls b and c in the presence of their pheophytins will be achieved either by extending the trichromatic method to a hexachromatic one, in which pheophytins as well as chlorophylls are estimated, or by using separation methods such as that de- veloped for chlorophyll c by Parsons (1963).

BRIAN Moss Department of Botany, University of Bristol, United Kingdom.

REFEERENCES

KAY, R. H. 1964. Experimental biology, mea- surement and analysis. Chapman and Hall, London. 416 p.

Moss, B. 1967. A spectrophotometric method for the estimation of percentage degradation of chlorophylls to pheo-pigments in extracts of algae. Limnol. Oceanog., 12: 335-340.

ODLUM, H. T., W. MCCONNELL, AND W. ABBOTT. 1958. The Chlorophyll 'A' of communities. Publ. Inst. Marine Sci. Texas, 5: 65-96.

PARSONS, T. R. 1963. A new method for the micro-determination of chlorophyll c in sea water. J. Marine Res., 21: 164-171.

PARSONS, T. R., AND J. D. H. STRICKLAND. 1963. Discussion of the spectrophotometric deter- mination of marine plant pigments with re- vised equations for ascertaining chlorophylls and carotenoids. J. Marine Res., 21: 155- 163.

RIcHAIRDS, F. A. wITH T. G. THOMPsON. 1952. The estimation and characterization of plank- ton populations by pigment analysis. II. A spectrophotometric method for the estimation of plankton pigments. J. Marine Res., 11: 156-172.

SMITH, J. H. C., AND A. BENITEZ. 1955. Chloro- phylls. Analysis in plant materials, p. 142- 196. In K. Paech and M. V. Tracey [eds.], Modem methods of plant analysis, v. 4. Springer-Verlag, Berlin.

STRICKLAND, J. D. H., AND T. R. PARSONS. 1965. A manual of sea water analysis. Bull. Fisheries Res. Board Can., 125, 2nd ed. 203 p.

TALLING, J. F., AND D. DRIVER. 1961. Some problems in the estimation of chlorophyll a in phytoplankton, p. 142-146. In M. S. Doty [ed.], Proc. Conf. Primary Production Mea- surement Marine Freshwater, Univ. Hawaii. U.S. Atomic Energy Commission Publ. TID 7633.

VERNON, L. P. 1960. Spectrophotometric deter- mination of chlorophylls and pheophytins in plant extracts. Anal. Chem., 32: 1144-1150.

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