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Chlorophyll-a determination with ethanol – a critical test

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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, 1992 standard, which prescribes 90% (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 from 20 to 0%. The extraction procedure was verified by measuring the amount of retained water after using both water and oil pumps for filtering 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 with 90% ethanol were compared to those obtained with methanol and acetone. On the basis of the calculated error level, improvements to the ISO: 10260, 1992 standard method have been suggested.

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References

  • Ballschmitter K. & J. J. Katz, 1972. Chlorophyll–Chlorophyll and Chlorophyll–water interactions in the solid state. BBA 256 (46277): 307–327.

    Google Scholar 

  • Balogh, K., A. Bothàr, K. T. Kiss & L. Vörös, 1994. Bacterio-, phyto-, lankton of the River Danube (Hungary). Verh. int. Ver. Limnol. 25: 1692–1694.

    Google Scholar 

  • Bernhardt, H. & J. Clasen, 1991. Flocculation of micro-organisms. J. Water SRT – Aqua 40: 76–87.

    Google Scholar 

  • Cattaneo, A., 1983. Grazing on epiphytes. Limnol. Oceanogr. 28: 121–132.

    Google Scholar 

  • Clarke, R. H., 1982. The chlorophyll triple state and the structure of chlorophyll aggregates. In Fong, F. K. (ed.), Light Reaction Path of Photosynthesis. Springer – Verlag Berlin Heidelberg New York: 196–233.

    Google Scholar 

  • Codd, G. A., C. Edwards, K. A. Beattle, W. M. Barr & G. J. Gunn, 1992. Fatal attraction to Cyanobacteria. Nature Volume 359: 110–111.

    Google Scholar 

  • Fong, K. F., 1975. Ester and keto carbonyl linkages in Chlorophyll a, Prochlorophyll a and Protochlorophyll a. J. am. Chem. Soc. 97: 23–6890.

    Google Scholar 

  • Fong, K. F. & V. J. Koester, 1975. Bonding interactions in anhydrous and hydrated Chlorophyll a. J. am. Chem. Soc. 97: 23–6888.

    Google Scholar 

  • Halling-Sorensen, B., 2000. Algal toxicity of antibacterial agents used in intensive farming. Chemosphere 40: 731–739.

    Google Scholar 

  • Horwitz, W. (ed.), 1955. Official Methods of Analysis of the Association of Agricultural Chemists 1955. Association of Official Agricultural Chemists, Washington: 939 pp.

    Google Scholar 

  • ISO 10260, 1992.Water quality, measurement of biochem. parameters; spectrometric determination of the chlorophyll-a concentration. Beuth Verlag GmbH Berlin-Vien – Zürich.

  • Iwamura, T., H. Nagai & S. Ichimura, 1970. Improved methods for determinig contents of chlyll, protein, ribonucleic acid, and dezoxyribonucleic acid in planctonic populations. Int. Rev. ges. Hydrobiol. 55/1: 131–147.

    Google Scholar 

  • Jeffrey, S. W. & G. F. Humphrey, 1975. New spectrometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanzen (BPP) Bd 167: 191–194.

    Google Scholar 

  • Jewson, D. H., G. H. Rippley & W. K. Gilmore, 1981. Loss rates from sedimentation, parasitism and grazing during the growth, nutrient limitation and dormancy of a diatom crop. Limnol. Oceanogr. 26: 1045–1056.

    Google Scholar 

  • Jones, R. J., 1995. The horizontal distribution of plankton in a deep oligotrophic lake – Loch Ness, Scotland. Freshwat. Biol. 33: 161–170.

    Google Scholar 

  • Jonsson, G. S., 1987. The depth-distribution and biomass of epilithic periphytonin Lake Thingvallavatn, Iceland. Arch. Hidrobiol. 108: 531–547.

    Google Scholar 

  • Kataura, K., S. Koseki, H. Ogawa, F. Yamazaki & Y. Tateno, 2000. Characteristics of ethanol-extracted chlorophyll-related compounds from salted brown seaweed, Konbu Laminaria japonica and its use on seasoning extractive of Konbu. Nippon Suisan Gakk 66: 104–109.

    Google Scholar 

  • Kiss, K. T., 1996. Diurnal changes of planctonic diatoms in the River Danube near Budapest (Hungary). Algological Studies 80 = Arch. Hidrobiol. Suppl. 112: 113–122.

    Google Scholar 

  • Kiss, K. T. & S. I. Genkal, 1993. Winter blooms of centric diatoms in the River Danube and in its side-arms near Budapest (Hungary). Hydrobiologia 269/270: 317–325.

    Google Scholar 

  • Kiss, K. T., J. Nosek & L. Kremmer, 1998. Diurnal change of phytoplankton, some physical and chemical components in the River Danube near Budapest (Hungary). Verh. int. Ver. Limnol. 26: 1041–1044.

    Google Scholar 

  • Lean, D. R. S. & F. R. Pick, 1981. Photosynthetic response to nutrient enrichment: a test for nutrient limitation. Limnol. Oceanogr. 26: 1001–1019.

    Google Scholar 

  • Levasseur, M. P., A. Thompson & P. J. Harrison, 1993. Physiological acclimation of marine phytoplankton to different nitrogen sources. J. Phycol. 29: 587–595.

    Google Scholar 

  • Lichtenthaler, H. K. 1987. Chlorophylls and caroteniods: Pigments of photosynthetic biomembranes. Methods in Enzymol. Volume 148: 350–382.

    Google Scholar 

  • Lund, J. W. G., G. C. Kipling & E. D. Lecreen, 1958. The inverted microscope method of estimating algal number and the statistical basis of estimation by counting. Hydrobiologia 11: 143–170.

    Google Scholar 

  • Mukherjee, T., A. V. Sapre & J. P. Mittal, 1978. On the nature of Chlorophyll a in aquaeous micellar systems. Photochem. Photobiol. V:28. 95.

    Google Scholar 

  • Pantecost, A. 1991. Algal and bryophyte flora of a Yorkshire (U.K.) hill stream: a comparative study approach using biovolume estimations. Arc. Hydrobiol. 121: 181–201.

    Google Scholar 

  • Porra, R. J., W. A. Thompson & P. E. Kriedman, 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying Chlorophylls a and b extracted with four different solvents: verification of concentration of Chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975: 384–394.

    Google Scholar 

  • Robinson, C. T. & S. R. Rushforth, 1987. Effect of physical disturbance and canopy cover on attached diatom community structure in an Idaho stream. Hydrobiologia 154: 49–59.

    Google Scholar 

  • Rosen, B. H. & R. L. Lowe, 1984. Physiological and ultrastructural responses of Cyclotella meneghiniana (Bacillariophyta) to light intensity and nutrient limitation. J. Phycol. 20: 173–183.

    Google Scholar 

  • Seely G. R. & R. G. Jensen, 1965. Effect of solvent on the spectrum of Chlorophyll. Spectrochimica Acta 12: 1935–1845.

    Google Scholar 

  • Shafik, H. M., S. Herodek, L. Vörös, M. Présing & K. T. Kiss, 1997. Growth of Cyclotella meneghiniana Kutz. I. Effect of temperature, light and low rate nutrient supply. Annls. Limnol. 33: 139–147.

    Google Scholar 

  • Skidmore, R., S. C. Maberly & B. A. Whitton, 1998. Patterns of spatial and temporal variation in phytoplankton chlorophyll a in the River Tent and its tributaries. Sci. Tot. Environ. 210/211: 357–365.

    Google Scholar 

  • Slatkin, D. N., R. D. Stoner, W. H. Adams, J. H. Kycia & H. W. Siegelman, 1983. Atypical pulmonary thrombosis caused by a toxic cyanobacterial peptide. Science 220: 1383–1385.

    Google Scholar 

  • Stauber, J. L. & S. W. Jeffrey, 1988. Photosynthetic pigments in fifty-one species of marine diatoms. J. Phycol. 24: 158–172.

    Google Scholar 

  • Standard Methods for the Examination of Water and Wastewater 18th Edition, 1992. Publ. Office: American Public Health Association, Washington D. C., ISBN 0-87553-207-1.

  • Sumner, W. T. & S. G. Fisher, 1979. Periphyton production in Fort River, Massachusetts. Freshwat. Biol. 9: 205–212.

    Google Scholar 

  • Talling, J. F. 1993. Comparative seasonal changes and inter-annual variability and stability in a 26-year record of total phytoplankton biomas sin four English lake basins. Hydrobiologia 268: 65–98.

    Google Scholar 

  • Thompson, R. C.,M. L. Tobin, S. L. Hawkins & T. A. Norton, 1999. Problems in extraction and spectrophotometric determination of chlorophyll from epilithic microbial biofilms: towards a standard method. J. mar. biol. Ass. U.K. 79: 551–558.

    Google Scholar 

  • Utermöhl, H., 1958. Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Mitt. int. Ver. Limnol. 9: 1–38.

    Google Scholar 

  • Vladkova, R., 2000. Chlorophyll-a self-assembly in polar solventwater mixture. Photocem. Photobiol. 71: 71–83.

    Google Scholar 

  • Vörös, L. & J. Padisák, 1991. Phytoplankton biomass and Chlorophyll-a in some shallow lakes in central Europe. Hydrobiologia 215: 111–119.

    Google Scholar 

  • Wellburn, A. R., 1994. The spectral determination of chlorophylls a and b as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144. 307–313.

    Google Scholar 

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Authors and Affiliations

  1. Doctoral School, Eötvös Loránd University of Science, Pázmány Péter allee 1/A, Budapest, H-1117, Hungary

    Éva Pápista

  2. Department of Microbiology, Eötvös Loránd University of Science, Pázmány Péter allee 1/C, Budapest, H-1117, Hungary

    Éva Ács

  3. Department of Plant Anatomy, Eötvös Loránd University of Science, Pázmány Péter allee 1/C, Budapest, H-1117, Hungary

    Béla Böddi

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  1. Éva Pápista
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  2. Éva Ács
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  3. Béla Böddi
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Correspondence to Béla Böddi.

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Pápista, É., Ács, É. & Böddi, B. Chlorophyll-a determination with ethanol – a critical test. Hydrobiologia 485, 191–198 (2002). https://doi.org/10.1023/A:1021329602685

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