SUMMARY: Concentrations of chlorophyll a and pheopigments, considered an index of the phytoplankton standing stock, are measured by fluorometry after particulate sample concentration by vacuum filtration and extraction in 100% acetone.
Historically chlorophyll a (chl a) has been used as a measure of phytoplankton standing stock. Also, when related to photosynthetic carbon production, the concentration of chl a and distribution in the water column allows one to estimate an index of the efficiency of phytoplankton in harvesting light.
Fluorescence is produced by atoms or molecules that, under exited state, return to their state of minimum energy (or ground state) by losing energy in the form of light. The absorption of blue light by chlorophylls and pheopigments produces emission of red light. In a sample, the fraction of light transformed is proportional to the amount of pigment. Hence, by knowing the amount of incident light and by measuring the light emitted at a second wavelength, it is possible to estimate the concentration of these pigments in the sample.
|2.1.||Seawater samples are collected from 5, 25, 45, 60, 75, 85, 95, 105, 115, 125, 150, 175 and 200 m using 12-liter Niskin bottles attached to the rosette sampler and from the primary productivity cast (see Chapter 14) using Go-Flo bottles attached to a Kevlar line.|
|2.2.||A 500 ml sample from each bottle is collected in clean 4-liter polyethylene bottles and stored in the dark. The polyethylene bottles are rinsed three times with 100-200 ml of sample before the collection of the final sample.|
|2.3.||Filtration and storage
|3.1.||A chl a standard stock is prepared every four months using commercially-available chl a (SIGMA) and 100% acetone. The stock is wrapped in aluminum foil and stored at -20 °C.|
|3.2.||To determine the concentration of the stock, a 50 ml sample is brought to room temperature avoiding exposure to the light. An absorbance spectrum from 350-750 nm is obtained using a scanning spectrophotometer and the concentration is calculated considering an extinction coefficient of 88.15 (l g-1 cm-1) for the absorbance at 662 nm.|
|3.3.||Using the same stock sample, three dilutions (1/10 each) are prepared gravimetrically using 100% acetone and read in the fluorometer. During this step, chl a must be kept under dim light.|
|3.4.||A calibration of the fluorometer is performed every 6 months following the procedure described by Strickland and Parsons (1972).|
|4.1.||Concentrations of chl a and pheopigments are calculated using
the following equations:
chl a = (T/(T-1))*(Rb-Ra)*Fd*volex/volfilt
phaeo = (T/(T-1))*((T*Ra)-Rb)*Fd*volex/volfilt
Niskin or Go-Flo bottles and rosette/CTD unit
fluorometer (Turner model 111)
spectrophotometer (Varian model DMS-100S)
HCl (1 M)
Strickland, J. D. H. and T. R. Parsons. 1972. A Practical Handbook of Seawater Analysis, Fisheries Research Board of Canada, 167 pp.