叶绿素a的测量-乙醇提取法

Hydrobiologia485:191–198,2002.

©2002Kluwer Academic Publishers.Printed in the Netherlands.

191 Chlorophyll-a determination with ethanol–a critical test

´Eva P´a pista1,´Eva´Acs2&B´e la Böddi3,∗

1Eötvös Lor´a nd University of Science,Doctoral School,P´a zm´a ny P´e ter allee1/A,Budapest H-1117,Hungary

2Eötvös Lor´a nd University of Science,Department of Microbiology,P´a zm´a ny P´e ter allee1/C Budapest H-1117, Hungary

3Eötvös Lor´a nd University of Science,Department of Plant Anatomy,P´a zm´a ny P´e ter allee1/C,Budapest

H-1117,Hungary

Tel:12660240;E-mail:bbfotos@ludens.elte.hu

(∗Author for correspondence)

Received2May2001;in revised form30August2002;accepted20August2002

Key words:algae,chlorophyll-a determination,ethanol,ISO standard10260(1992)

Abstract

Chlorophyll-a content is widely used as an indicator of the quality of freshwater bodies.Quantification of chlorophyll-a is a routine procedure in the test laboratories of water works,and in research laboratories.Although attempts have been made to standardise the measurement procedure,there are nonetheless many procedures currently in use.This work is focused on a careful re-examination of the ISO:10260,1992standard,which prescribes90%(v/v)ethanol for chlorophyll extraction and measurement.Chlorophyll contents of cultures of the cyanobacterium Synechococcus elongatus Nägeli and the chlorophyte Scenedesmus acutus Meyen were determined by means of a series of concentrations of ethanol/water mixtures which were employed as extracting agents–the water content was gradually decreased from20to0%.The extraction procedure was verified by measuring the amount of retained water after using both water and oil pumps forfiltering the samples.The spectroscopic effects of the presence of water were studied and the molecular background of these spectral phenomena is discussed.The extraction yields obtained with90%ethanol were compared to those obtained with methanol and acetone.On the basis of the calculated error level,improvements to the ISO:10260,1992standard method have been suggested.

Introduction

The chlorophyll(Chl)content of freshwater bodies is a widely accepted indicator of water quality.Research projects on periphyton(Cattaneo,1983;Jonsson, 1987;Robinson&Rushforth,1987;Pantecost,1991) or phytoplankton(Kiss&Genkal,1993;Balogh et al., 1995;Jones,1995;Kiss,1996;Shafik et al.,1997; Skidmore et al.,1998;Kiss et al.,1998)use these characteristics to describe the trophic state(Sumner &Fisher,1979;Vörös&Padisák,1991;Talling, 1993)of the studied system.However,the identific-ation of the alga species,the knowledge of the algal cell number,or the physiological state of cells may also be important in providing a true picture of the water quality or trophic state.A combination of Chl determination and consideration of these other factors may provide an improvement in the reliability and ac-curacy of water quality estimation.Utermöhl(1958) developed a method to determine the individual num-ber of algae with an inverted microscope and Lund et al.(1958)described a procedure to estimate the accuracy and limitations of Utermöhl’s method.

If certain taxa are in developing or degrading stages in the studied populations,consideration of the factors above is essential,since certain species produce toxins harmful to both water animals and hu-man(Slatkin et al.,1983;Codd et al.,1992).It has been established that the presence of algae and thus the Chl content indicate the concentration of certain chemicals or the appearance of toxins in the drinking water(Bernhardt&Clasen,1991).Thus,considera-