WATER QUALITY: Analytical Methods--Biological March 15, 1976 QUALITY OF WATER BRANCH TECHNICAL MEMORANDUM NO. 76.14 Subject: WATER QUALITY: Analytical Methods--Biological Attached is one copy each of the following methods that were developed by W. Thomas Shoaf and Bruce W. Lium of the Biological Methods Development Project in Doraville, Georgia: (l) Determination of Chlorophyll from Phytoplankton (Chromatographic Method) (2) Determination of Chlorophyll from Periphyton (Chromatographic Method) (3) Determination of Biomass/Chlorophyll Ratio for Plankton (4) Determination of Biomass/Chlorophyll Ratio for Periphyton. The basic chromatographic procedure used in all four methods for the determination of chlorophyll is an advance of the state-of- the-art and a significant improvement over the spectrophotometric methods specified in Methods for Collection and Analysis of Aquatic Biological and Microbiological Samples (Slack and others, 1973, TWRI, Bk. 5, Ch. A4, p. 57-64). The reasons for the improvement are as follows: (l) A markedly more efficient extraction procedure (that is, the use of dimethyl sulfoxide, DMSO) for removing chlorophyll from green algae is used. With some green algae, from 2 to 60 times more chlorophyll is extracted as with the previous technique (that is, acetone); this resulted in a significant underestimation when green algae were the predominant forms present. (2) Chlorophyll degradation products (pheophytins), often present in substantial concentrations with respect to chlorophyll concentration, interfered directly with the previous method and in many cases resulted in an overestimation of chlorophyll. There are no degradation product interferences with the new method. (3) Turbidity sometimes caused an overestimation of chlorophyll with the previous method; it has no effect on the new method. (4) The addition of magnesium carbonate (previous method) was of questionable use, because experiments in Doraville have shown that it has no effect either in aiding the retention of algae during filtration or in the preservation of chlorophyll. (5) The enclosed methods for determination of chlorophyll give two to three orders of magnitude greater sensitivity than the previous methods because they allow the use of more sensitive fluorometric instrumentation. The capability for analyzing a limited number of samples using the new methods will be available upon request from the Atlanta Central Water Quality Laboratory beginning about May 1. The analytical costs until FY 77, and lab codes for each determination, are as follows: Lab Code Determination Cost PHYTOPLANKTON 618 Chlorophyll a ug/l 15 00 619 Chlorophyll b ug/l 15.00 620 Biomass (dry wt.) mg/l 12.19 621 Biomass (ash wt.) mg/l 12.19 622 Biomass/Pigment Ratio* Calculation PERIPHYTON 623 Chlorophyll a mg/m2 15 00 624 Chlorophyll b mg/m2 15.00 625 Biomass (dry wt.) mg/m2 12 19 626 Biomass (ash wt.) mg/m2 12.19 627 Biomass/Pigment Ratio* Calculation *Dimensionless No STORET parameter codes are available at this time. The attached methods have been approved by the Quality of Water Branch as provisional methods. The chromatographic methods will become the standard methods in the Atlanta Central Laboratory on October 1, 1976. After that date, the spectrophotometric methods will be available only upon special request for completion of ongoing projects. Because of the improved extraction efficiency and absence of interference from degradation products, comparison of data from the two type of methods may not be possible. Please distribute the attached methods to your personnel as appropriate. Questions or comments pertaining to the provisional methods should be directed to the Chief, Quality of Water Branch (Mail Stop 412, Reston, Virginia). Questions pertaining to analytical services should be directed to the Chief, Atlanta Central Water Quality Laboratory (WRD, Doraville, Georgia). R. J. Pickering Attachments WRD Distribution: A,B,FO-LS,PO