Chlorophyll a Measurements in Long Island Sound
STAC Meeting June 15, 2018 Jim Ammerman Science Coordinator, Long Island Sound Study, New England Interstate Water Pollution Control Commission (NEIWPCC) 2
Questions have arisen about measurements of Chlorophyll a made by different groups in LIS and what is meant by “Corrected Chlorophyll a ” 3
Structure of Chlorophyll a Molecule The chlorophyll a molecule, consisting of a porphyrin ring, a chelated magnesium molecule in the ring (purple), and a long hydrocarbon (phytol) “tail.” Model courtesy of Botany Online, University of Hamburg 4
Pathways of Chlorophyll Degradation 5
Extracted Chlorophyll a Methods 1. Absorbance measurements with spectrophotometer (w/or w/o acid additions*) 2. Fluorescence measurements with fluorometer (w/or w/o acid additions) 3. HPLC measurements with fluorescence detector (w/or w/o acid additions) *Note: Acid addition removes Mg, producing pheophytin a 6
EPA Chlorophyll a Method 445.0 (1) (In the Quality Assurance Plan for U. Connecticut’s Center for Environmental Sciences and Engineering or CESE) This method provides a procedure for low level determination of chlorophyll a (chl a ) and its magnesium free derivative, pheophytin a (pheo a ), in marine and freshwater phytoplankton using fluorescence detection. Phaeophorbides present in the sample are determined collectively as pheophytin a . For users primarily interested in chl a there is currently available a set of very narrow bandpass excitation and emission filters (Turner Designs, Sunnyvale, CA) that nearly eliminates the spectral interference caused by the presence of pheo a and chlorophyll b . 7
EPA Chlorophyll a Method 445.0 (2) (In the Quality Assurance Plan for U. Connecticut’s Center for Environmental Sciences and Engineering or CESE) The difference between the modified method and the conventional fluorometric method is that the equations used for the determination of chlor a without pheo a correction (uncorrected chlor a ), are used instead of the equations for "corrected chlor a ". This EPA laboratory has evaluated the modified filters and found the technique to be an acceptable alternative to the conventional fluorometric method using pheo a correction. (Note: This method will not provide a pheophytin a value, some may find pheophytin useful as a measure of grazing.) 8
LIS Chlorophyll a Measurements 1. CT DEEP has used the narrow bandpass filter method outlined above for decades to measure “corrected chlorophyll a ” without the acidification step (analysis done at U. Conn. CESE) 2. IEC-Prior to 2011, samples were sent to U. Conn. CESE. From 2011- 2016, samples were measured with a spectrophotometer before and after acidification. Since 2016, the same before and after acid measurements have been made with a fluorometer. 3. CT DEEP also appropriately “corrects” its CTD chlorophyll values with extracted chlorophyll measurements from the same day, this has also confused some 9
Very Limited Comparisons Suggest Some Differences Between CT DEEP and IEC Measurements at Adjacent Stations (EPA Staff) 10
Additional Information on Narrow Bandpass Filter Method 1. EPA: Evaluation of a New Fluorometric Technique that Uses Highly Selective Interference Filters for Measuring Chlorophyll a in the Presence of Chlorophyll b and Pheopigments (https://nepis.epa.gov/Exe/ZyPDF.cgi/9100PTUC.PDF?Dockey=9100 PTUC.PDF) 2. Turner Designs: Application Note: Non-Acidification Technique for Extracted Chlorophyll a Analysis (https://www.turnerdesigns.com/t2/doc/appnotes/S-0013.pdf) 11
Questions? 12
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