A critical comparison of methods for the determination of phytoplankton chlorophyll Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/n296x135h

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  • The concentration of chlorophyll in natural bodies of water is commonly determined as a means to rapidly estimate the phytoplankton biomass. The literature gives numerous warnings, however, as to the problems involved with accurately determining chlorophyll concentrations. The author's work at Crater Lake, Oregon enticed him to explore critically the spectrometric methods for determining chlorophyll. Four spectrometric methods for the determination of chlorophyll have been investigated. These are the spectrophotometric method, the 'in vitro' fluorometric method, the 'in vivo' fluorometric method and the 'in situ' fluorometric method using fiber optic cables (remote fiber fluorometry). The spectrophotometric trichromatic and monochromatic methods depend on absorption measurements made with a spectrophotometer. The spectral bandpass of the spectrophotometer is a critical variable in the determination of chlorophyll. A spectral bandpass of 2.0 nm has been suggested and shown to be adequate to measure the concentrations of chlorophyll-a. The chlorophyll concentration determined is 15% and 36% too low with spectral bandpasses of 10 and 20 nm, respectively. Increasing the spectrophotometric cell pathlength from 1.0 to 5.0 cm improves the detection limit of the method by a factor of 5. With a 1-cm pathlength cell, the detection limit for chlorophyll-a is 34 µg/L in an extract or 0.34 µg/L in lake water with a concentration factor of 100. Of the fluorometric methods studied, the 'in vitro' uncorrected fluorometric method was shown to be the most precise and to provide the lowest detection limit (4 ng/L in an extract and 0.04 ng/L chlorophyll-a in lake water with a concentration factor of 100). The detection limits for the 'in vivo' and the enhanced 'in vivo' method (using DCMU) fluorometric methods are 5 and 3 ng/L, respectively. The effect of several variables in the sample preparation method for the spectrophotometric and 'in vitro' fluorometric methods were studied with samples of Cronemiller Lake water. No difference in filter retention efficiency at the 95% confidence level was observed when the Millipore HA membrane, S & S glass and Whatman Glass GF/F filters were compared with a solution of titanium dioxide or a natural phytoplankton sample. Following 65 days of storage at 0° C or 238 days of storage at 9° C, the chlorophyll concentration determined did not significantly change from that determined at the beginning of the study. The use of MgCO₃ did not change this condition. The 'in vivo' fluorometric technique, applied to water samples from Crater Lake, Oregon, was shown to be influenced by sample temperature and irradiance history. The addition of the herbicide DCMU to a sample has been reported to decrease the dependency of the fluorescence signal on temperature and irradiance history of the sample. This was shown not to be the case. A 10° C decrease of sample temperature resulted in an average 1.8% increase in sample fluorescence. Exposure of a set of samples to solar radiation decreased the fluorescence signal for chlorophyll in the samples. A period of great change in fluorescence signal was followed by an extended period of slower change. After 50 minutes of sample irradiation, the average fluorescence signal decreased over 50% relative to the original signal. A remote fiber fluorometer was constructed to investigate its use for the 'in situ' fluorometric determination of chlorophyll. Transmission characteristics of the fiber showed that light attenuation increased as the wavelength decreased. With a jig that held the excitation and emission fibers at varying distances and angles, it was found that maximum fluorescence signals were recorded as the fiber ends were moved as close as possible to each other and at an angle of about 10°. The 'in situ' detection limit for chlorophyll-a was determined to be 0.64 µg/L using 1-m excitation and emission fibers.
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