BS ISO 10260:1992

1.1 This International Standard specifies a method for the determination of the chlorophyll-a concentration. The procedure can be applied for phytoplankton in natural surface waters and for testing algal growth in bio-assays. Using appropriate sampling it can also be applied to phytobenthic communities (see annexA).

1.2 Other algal pigments such as chlorophyll-b and chlorophyll-c and some chlorophyll metabolites do not contribute to the determination. Phaeopigments may be determined semiquantitatively, to correct for interference with chlorophyll-a determination and to indicate the portion of inactive algal biomass.

1.3 Chlorophyll is sensitive to light and oxygen, especially when it is extracted. To avoid oxidative and photochemical destruction, the samples shall not be exposed to bright light or air. Homogenization of the sample may in some cases increase the extraction efficiency.

1.4 The extraction procedure with ethanol involves heating to 75°C for 5min to inactivate chlorophyllase and accelerate the lysis of pigments. Storage of extracts (except filters containing suspended matter) prior to photometric measurement should be kept to a minimum, but is possible up to 3d under refrigeration at 4°C. Storage of extracts at less than−25°C is possible for at least 30d.

1.5 Even though the procedure involves filtration or centrifugation to clarify the final extract, a slight turbidity may remain. The acidification step may also cause turbidity. Therefore, the absorbance measured at 665nm has to be corrected for turbidity by substracting the absorbance measured at 750nm.

1.6 The pigment of certain rarely occurring phototrophic bacteria (e.g. Chlorobium) interferes with the determination of chlorophyll-a concentration [1]. The contribution of chlorophyll-b and chlorophyll-c to the absorbance at 665nm is negligible [2].