Water quality -- Measurement of biochemical parameters -- Spectrometric determination of the chlorophyll-a concentration

Document Number: NL ISO 10260 : 2010
Sector: Food Technology
TC: NL TC 147
ICS: 13.060.50
1.1 This International Standard specifies a method for the determination of the chlorophyll-a concen- tration. The procedure tan be applied for phytoplankton in natura1 surface waters and for testing algal growth in bio-assays. Using appropriate sampling it tan also be applied to phytobenthic communities (see annex A). 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 Iight 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 ex- traction efficiency. 1.4 The extraction procedure with ethanol involves heating to 75 OC for 5 min to inactivate chlorophyllase and accelerate the lysis of Pigments. Storage of extracts (except filters containing sus- pended matter) Prior to photometric measurement should be kept to a minimum, but is possible up to 3 d under refrigeration at 4 OC. Storage of extracts at less than - 25 OC is possible for at least 30 d. 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 665 nm has to be corrected for turbidity by substracting the absorbance measured at 750 nm. 1.6 The Pigment of certain rarely occurring phototrophic bacteria (e.g. Chlorobium) interferes with the determination of chlorophyll-a concen- tration [l]. The contribution of chlorophyll-b and chlorophyll-c to the absorbance at 665 nm is negli- gible [2].