PHYTOPLANKTON CHLOROPHYLL

Chlorophyll measurements were performed by both monochromatic and by trichromatic spectrophotometric methods according to Lorenzen (1967) and Jeffrey and Humphrey (1975) respectively.

Sampling

Chlorophyll samples were taken from the same water bottles used for phytoplankton and primary production analyses at 0 m, 5 m, 10 m, 15 m, 20 m and 30 m depths. Two dm≥ was filtered immediately after sampling in subdued light using a GF/f filter. Suction pressure did not exceed 3 x 104 Pa. The filtration time lasted a maximum of 30 minutes. The filters were drained under suction before removal from the filtration equipment and stored deep-frozen until extraction.

Extraction

The folded filter was placed in a graduated centrifuge tube with a proper stopper in order to be able to adjust the volume and to avoid evaporation:

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

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

The measurements were made immediately after centrifugation. The volume was adjusted before reading.

For spectrophotometric readings and calculations we followed the instructions of Lorenzen (1967) and Jeffrey and Humphrey (1975) respectively and some specifications of Edler (1979).

References

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.

Jeffrey, S.W. and G. Humphrey 1975. New spectrophotometric equations for determining chlorophyll a,b,c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanzen 167: 191-194.

Lorenzen, C.J. 1967. Determination of chlorophyll and pheo-pigments: spectrophotometric equations. Limnol. Oceanogr. 12: 343-346.