Annex C-4. Phytoplankton chlorophyll a

1. Introduction

Nutrient enrichment/eutrophication may give rise to increased phytoplankton biomass, increased frequency and duration of phytoplankton blooms and increased primary production. Chlorophyll-a is used as an estimate of phytoplankton biomass.

2. Purpose

The purposes for measuring chlorophyll-a concentration in the Baltic Sea Area are:

• to describe the spatial distribution and frequency of phytoplankton blooms;
• to describe temporal trends in phytoplankton biomass and in the frequency and duration of phytoplankton blooms.

3. Sampling strategy

Increased chlorophyll-a concentrations mainly occur in nutrient-enriched waters and in the deeper layers of stratified water. To describe the spatial distribution of chlorophyll-a concentrations, horizontal and vertical sampling at an appropriate number of stations is required. In order to describe the spatial distribution and surface coverage of phytoplankton blooms, regular spatial surveys could be carried out using ships of opportunity or aerial visual and remote sensing devices, accompanied by regular water sampling. In order to give exact information on the vertical distribution it is recommended to make profiles with a fluorometer.

Chlorophyll-a concentrations vary substantially during the growth season and may vary considerably from year to year as a consequence of many factors (e.g. meteorological/hydro-graphic conditions). Thus, it may be difficult to describe temporal trends. For this reason, sampling needs to cover the entire growth season. This leads to the possibility of assessing mean values for the spring season and for the entire growth season.

The sampling strategy for sea-truth measurements follows the general HELCOM strategy of shared ship cruise measurements on agreed stations in the Baltic Sea. The following text describes only ship cruise measurements of chlorophyll-a and phaeopigments.

4. Sampling

For the open sea, the standard sampling depths for chlorophyll-a are in the upper water column the same as for nutrients: 1 m, 5 m, 10 m, 15 m and 20 m. In CMP, the sample from 1 m or an integrated sample (1-10 m) could be analysed. Additional sample(s) should be obtained from chlorophyll maxima present at other depths. Such maxima are found using a profiling fluorometer/ CTD. Chlorophyll should also be analysed from the same sample used for phytoplankton and primary production analyses.

For ships-of-opportunity and helicopter sampling a single sample from the mixed surface layer can be taken.

5. Storage of water samples

It is important that the water is filtered immediately after sampling.

6. Volume determination

6.1 Spectrophotometric determination

Due to the seasonal variations of chlorophyll-a concentrations in the Baltic Sea area the sample volumes have to be optimized to fit the volume of the extraction solvent and the cuvette length:
- at a concentration of 0.1 mg x m^-3:

10 cm³ extraction solvent and 5 cm cuvette length needs 1200 cm³ of water to be filtered to get a spectrophotometric absorbency of about 0.05.
- at a concentration of 1 mg x m^-3:

10 cm³ extraction solvent and 1 cm cuvette length needs 600 cm³ of water to be filtered to get a spectrophotometric absorbency of about 0.05.

6.2 Fluorometric determination

The sensitivity of the fluorometer is about ten times higher compared to the spectrophotometer. Thus, the required volume could be reduced ten times.

7. Filtration

- The samples shall be filtered in subdued light.
- The filtration should be carried out immediately after sampling.
- Filter to be used: GF/F
- Suction pressure must not exceed 3 x 10⁴ Pa.
- Filtration time must not exceed 30 min for the volumes needed for the spectrophotometric method and 3 min for the fluorometric method.

8. Drying of filters

The filter should be drained under suction before removal from the filtration equipment. The filter should be dried in darkness at room temperature before extraction.

9. Storage of filters

Extraction and analysis should be done without delay. If necessary, filters may be stored deep-frozen up to 1 month before extraction and analysis (Jeffrey et al., 1997).

10. Extraction

All work with the chlorophyll extract shall be carried out in subdued or green light.

After the filtration and drying, the folded filter is placed in a graduated centrifuge tube with proper stopper, to be able to adjust the volume and to avoid evaporation.

- 96% ethanol should be used as solvent.
- Extraction volume: 10 cm³ added with a calibrated dispenser/pipette, smaller amount could be used to get the optimal concentration (see Chapter 6.1).
- Extraction time: 6-24 hours at room temperature.
- Mixing: Constant or by shaking the tubes a few times during the extraction time.
- The extraction tube should be closed immediately after adding ethanol and should be kept tightly closed during extraction, storage and centrifugation.

11. Centrifugation

Using spectrophotometric measurements centrifugation is necessary.

If necessary, the extract volume is adjusted to 10 cm³ before centrifugation. The stoppered centrifuge tube is shaken vigorously to get a homogeneous distribution of the chlorophyll in the extraction solvent.

The sample is centrifuged for 10-20 minutes at about 10,000 m s^-2, in order to reduce the spectrophotometric blank reading (750 nm) which should not exceed 0.005 for a 1 cm cuvette.

12. Storage of extract

The measurements shall be made immediately after centrifugation. If this is not possible the extract may be stored in a deep freezer (-20^oC) for no more than 24 hours. The volume must be adjusted before reading.

13. Chlorophyll-a measurement procedure

The measurements of chlorophyll-a may either be made by a spectrophotometer or a fluorometer. The measurements of phaeopigments are tentative and shall only be made using a fluorometer.

13.1 Spectrophotometric readings

The spectrophotometer should be calibrated in terms of absorbency and wave-length at least once a year.

- Bandwidth: 2 nm
- Wavelength: 750 and 663-665 nm (at the peak)
- Reference: 96% ethanol
- Cuvette: flexible, due to concentration (note the length)

The zero correction of the baseline and a cell to cell blank must be done in order to define the zeropoint of the absorbency in the extraction medium.

Calculations

To calculate the chlorophyll-a content using the spectrophotometric technique, the following equation should be used:

e = volume of ethanol, cm³

A(665 k) = absorbency at 665 nm (the peak) minus the absorbency at 750 nm after correction by the cell-to-cell blank
l = length of cuvette, cm
V = water volume filtered, dm³
83 = absorption coefficient in 96% ethanol

The volume of chlorophyll sample, volume of ethanol and the length of cell (cuvette) must be chosen to give absorbency at 663-665 nm of 0.05-0.8, i.e. the optimum range of the spectrophotometer.

13.2 Fluorometric reading

It is necessary to make sure that the concentration of the extraction is at the optimum range of the instrument used. Synthetic detergents should be avoided when cleaning the cuvettes, as they may interfere with the fluorescence.

• Excitation setting: 425-430 nm
• Emission setting: 663-665 nm

Filter fluorometers must be equipped with a blue lamp corresponding to GE F4T5B, red-sensitive photomultiplier and primary filter corresponding to Corning 5-60 and secondary filter corresponding to Corning 2-64.

The fluorometer gives relative values which have to be converted to chlorophyll-a concentration using a calibration factor. The calibration factor is determined from the spectrophotometric reading or directly from fluorometric reading, in both cases using certified reference material.

For the measurement of phaeopigment the extract shall be acidified with 1 M HCl (0.06 cm³ to 5 cm³ of extract) after the first reading. 0.5-3 minute after acidification, a new reading should be made.

Calculations

Use the equation:

Chl.-a (mgm^-3) = RfseV^-1

R = fluorescence reading
f = calibration factor
s = slit correction
e = volume of ethanol (cm³)
V = volume of filtered water (dm³)

The calibration factor is determined as follows:

f = KR ^-1 Ve^-1

K = concentration of chlorophyll-a (mgm^-3) determined spectrophotometrically as described by Arvola, 1981.

When phaeopigment is to be calculated use the equation:

Phaeopigment (mg.m^-3) = f_a ((r R_a) - R) s e V^-1

R_a = fluorescence reading after acidification
r = ration R/R_a obtained from an extract free from phaeopigment
f_a = f r (r^-1)
f, R, s, e, V = see above

14. Analytical quality assurance

The general aspects of quality assurrance for chlorophyll a are covered under Part B, B.5.3.1.

15. Data reporting

For data reporting ICES environmental data reporting format should be used.

References and additional literature

Arvola, L., 1981. Spectrophotometric determination of chlorophyll a and phaeopigments in ethanol extraction. Ann. Bot. Fennici, 18, 221-227.

Edler, L., 1979. Recommendations for marine biological studies in the Baltic Sea: Phytoplankton and chlorophyll. Working Group 9, The Baltic Marine Biologists, publication No. 5, 38 pp.

HELCOM, 1988. Guidelines for the Baltic Monitoring Programme for the Third Stage. Part D. Biological Determinands.

Jeffrey, S.W., Mantoura, R.F.C., Wright, S.W., 1997. Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO Publishing, Paris, 661 pp.

Lindahl, O., 1986. A dividable hose for phytoplankton sampling. In Report of the ICES Working Group on Exceptional Algal Blooms, Hirtshals, Denmark, 17-19 March, 1986. ICES, C.M. 1986/L:26.

Strickland, J.D.H. and Parsons, T.R., 1968. A practical handbook of seawater analysis. Fish. Res. Board of Canada, Bulletin 169, Ottawa.

UNESCO, 1994. Protocols for the Joint Global Ocean Flux Study (JGOFS). Core Measurements. IOC Manuals and Guides 29: 179 pp.

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