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Chlorophyll a

Soil & Water Conservation Society of Metro Halifax (SWCSMH)

December 26, 2015

(Click on the picture to launch a PDF file for educational purposes including acknowledgements)

Introduction

(Vollenweider and Kerekes, 1982)

Chlorophyll a is considered the principal variable to use as a trophic state indicator. There is generally a good agreement between planktonic primary production and algal biomass, and algal biomass is an excellent trophic state indicator. Furthermore, algal biomass is associated with the visible symptoms of eutrophication, and it is usually the cause of the practical problems resulting from eutrophication. Chlorophyll a is relatively easy to measure compared to algal biomass. One serious weakness of the use of chlorophyll a is the great variability of cellular chlorophyll content (0.1 to 9.7 % of fresh algal weight) depending on algal species. A great variability in individual cases can be expected, either seasonally or on an annual basis due to a species composition, light conditions and nutrient (particularly nitrogen) availability.

The methods used for chlorophyll a determination are usually corrected for phaeophytin, the pigment fraction which is not active in photosynthesis. It could be argued, however, that the uncorrected (for phaeophytin) chlorophyll data used by Sakamoto (1966) might be better for chlorophyll total phosphorus correlations, since phaeophytin also contains phosphorus.

Chlorophyll a is to be measured within the euphotic zone. Simply, the euphotic zone is defined as the depth at which the light intensity of the photosynthetically active spectrum (400-700 nm) equals 1% of the subsurface light intensity (from photometric measurements with a Spherical Quantum Sensor and a DataLogger). It is desirable to use a spherical quantum sensor (4π type). Where this information is not available, a Secchi disc (SD) reading (in meters) in which Z_e= 2.5 x SD may be used. For coloured (dystrophic lakes), the factor will be lot lower.

Measurement of photosynthetic pigments in freshwaters

(Marker et al, 1980)

Sampling, transport and storage:

The volume of water filtered should be adjusted to take account of the concentration of algae. Water samples should be kept cool and dark and processed without delay. Storage in a refrigerator (5 ^oC) for some hours (e.g., overnight) may be possible. Storage of water samples for longer periods depends both on the phytoplankton composition (e.g., populations dominated by oscillatoria agardhii may be stored up to seven days at 5 ^oC) and on the concentration of the phytoplankton. Eutrophic waters with high concentrations of phytoplankton populations are often less suitable for storage than populations of low density.

Potential for grave errors:

Lyophilization and deep-freezing of samples can lead to pigment losses. However, the deep freezing of damp samples has been reported as satisfactory. The losses of pigments reported in the literature may be due to MgCO₃ added to the filters before freezing; algal-MgCO₃ aggregates are thought to retain cholorophyll during subsequent extractions.

MgCO₃ is frequently used to increase the retention efficiency of the glass-fibre filters and to avoid degradation of the chlorophylls (Strickland and Parsons, 1968). The use of MgCO3 has, however, been criticized on the grounds of differences in extraction efficiencies after freezing the filters. Severe errors may be introduced by the adsorption of chlorophyllides and phaeophorbides into MgCO₃. However, several investigators did not find any significant differences between chlorophyll concentrations obtained from filters with or without MgCO₃.

Storage of filters in acetone or ethanol may be possible for a few days at 4 oC in the dark, but Riemann (1976) reported a loss of 22% and a doubling of phaeopigments after 20h storage in methanol.

It is clear, however, that if storage is required for more than a few hours, the effects on any storage combination should be carefully checked.

Acidification procedures:

In selecting acidification procedures the following criteria must be satisfied: Conditions leading to the formation of dications of phaeophytin should be avoided. Conditions leading to the breakdown of expoxidic carotenoids must be avoided. Conversion of chl.a-phaeophytin a should be as rapid as possible.

Spectrophotometric methods:

The absorbance of a pigment extract is best measured in a spectrophotometer with a grid-monochromator because the optics of prismatic monochromators are inferior. Accurate wavelength setting is essential. Inaccuracies of 5 nm can lead to errors of 20-30%. A deviation of only 1 nm may cause an eror of 15% in the measurement of chlorophyll b in a pigment extract. The photometer must be adjusted frequently by means of the hydrogen line at 656.3 nm.

Differences in absorbance of spectrophotometer cuvettes filled only with the reference solvent should be zero. Any difference should be used to adjust the absorbance of the sample. Any further difference in absorbance between the reference solvent and sample at 750 nm is assumed to be turbidity and should not, in any case, exceed 0.005. The absorbance at 665 nm should be in the range 0.2-0.8 and should not fall below 0.1.

Caution- differences in the procedures for marine and fresh waters!

HPLC (High-performance liquid chromatography) pigment analyses

Taxonomically diagnostic carotenoids and chlorophylls (Chl) and their representative major freshwater algal groups. High-performance liquid chromatography calibration equations were derived for each pigment using standards provided by the US Environmental Protection Agency (Cincinnati, Ohio). (Vinebrooke and Leavitt, 1999)
Pigment	Algal group
Chl a	All algae
Chl b	Chlorophytes, euglenophytes
Chl c	Chromophytes, cryptophytes^a
Alloxanthin	Cryptophytes
Canthaxanthin	Filamentous cyanobacteria
Diadinoxanthin	Chromophytes, euglenophytes
Diatoxanthin	Diatoms, few chromophytes
Fucoxanthin	Chromophytes
Lutein	Chlorophytes
Zeaxanthin	Cyanobacteria
Myxoxanthophyll	Colonial cyanobacteria
Scytonemin	Some filamentous cyanobacteria
Violaxanthin	Chlorophytes

^a Chl c₂ reported in some cryptophytes

An analytical procedure for measuring Chlorophyll "A" in freshwaters

General Discussion:

Chlorophyll is a common indicator of phytoplankton biomass. All green plants contain chlorophyll "a" and, for planktonic algae, it constitutes about 1 to 2% of the dry weight. Other pigments that occur in plankton algae are chlorophyll "b" and "c", xanthophylls, and carotenes. The presence or absence of various pigments is used, among other features, to separate the major algal groups.

The determination of chlorophylls by the trichromatic method is of questionable value. It tends to overestimate chlorophyll "a" when no correction is made for the presence of the degradation product, pheophytin "a". Chlorophyll "b" and "c" values are calculated from readings taken on the slope of the chlorophyll "a" curve and are unreliable. For routine work in fresh water, determination of chlorophyll "a" and pheophytin "a" by spectrophotometry is the most valuable technique.

Two methods for determining chlorophyll "a" in phytoplankton are available, the spectrophotometric and the fluorometric. Fluorometry is more sensitive than spectrophotometry, requires less sample, and can be used for in-vivo measurements.

Pheophytin "a", a common degradation product of chlorophyll "a", can interfere with the determination of chlorophyll "a" because it absorbs light and fluoresces in the same region of the spectrum as chlorophyll "a" and, if present, may cause errors in chlorophyll "a" values. It can be measured either by spectrophotometry or fluorometry, but in fresh water fluorometric measurement is unreliable. The fluorometric method was developed for marine work (chlorophyll "b" is undetectable in the open ocean) and depends on the absence of chlorophyll "b", because after acidification, the fluorescence emission of pheophytin "b" is coincident with that of pheophytin "a".

When measuring chlorophyll "a", measure also the concentration of pheophytin "a". The ratio of chlorophyll "a" to pheophytin "a" serves as a good indicator of the physiological condition of phytoplankton. When pheophytin "a" is measured by spectrophotometry, accurate acidification is required to avoid interference from accessory pigments present in some algae.

Chlorophyll "a" is used as an algal biomass indicator. Assuming that chlorophyll "a" constitutes, on the average, 1.5% of the dry weight of organic matter (ash-free weight) of algae, estimate the algal biomass by multiplying the chlorophyll "a" content by a factor of 67.

Conduct work with chlorophyll extracts in subdued light to avoid degradation. Use opaque containers or wrap with aluminum foil.

Scope and Application:

This method is applicable to drinking water and surface waters only.

Summary of Method:

The pigments are extracted from the plankton concentrate with aqueous acetone and the fluorescence of the extract is determined with a spectrophotofluorometer.

Principle of Method:

A water sample is filtered using a glass fiber filter to recover the plankton. Acetone is used to extract the chlorophyll "a". The sample is read before and after acidification using a spectrophotofluorometer and the chlorophyll "a" is calculated by comparison with known standards.

Sampling procedure, Preparation and Storage:

Samples should be filtered as soon as collected. If this is not possible, samples are to be filtered as asoon as they reach the lab or stored for as short a time as possible at 4�C.

Filtered samples should be capped and kept in a freezer until ready to proceed with the extraction.

At least 500 ml sample should be collected for analysis.

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