The basic method of Holm-Hansen et al. (1965) and Strickland and Parsons (1972), with the modifications noted below, was used to analyze chlorophyll-a samples from the beginning of the LTER dataset in 1993 . Notes on the method and its modifications follow. Lake water samples were collected at specific depths with a five-liter Niskin bottle during normal LTER limnological sampling. Sub-samples were decanted into an amber one-liter Nalgene bottle. Two-100 mL or 200 mL sub-samples were taken from the one-liter amber Nalgene bottle. In a darkened environment, each sample was filtered through a combusted (475C for 4 hours) Whatman 25 mm GF/F using a bell jar apparatus. The filter was folded in half (organic material inside), placed in a glassine envelope, covered with aluminum foil, and frozen immediately for later analysis in McMurdo. The filtrate was collected for nutrient and dissolved organic carbon analyses. All of the following laboratory methods were performed in a darkened environment. In McMurdo, a chlorophyll-a stock concentrate was prepared by dissolving 1 mg of chlorophyll-a standard (Sigma, Anacystic nidulans) in a 100 ml volumetric flask and diluting it to mark with 90% acetone (~10,000 micrograms per liter chlorophyll-a). A Beckman DU-640 UV/vis spectrophotometer was used to determine the actual chlorophyll-a concentration of the stock concentrate and the concentration of the stock dilutions. The absorbance of the stock concentrate was measured at 665 nm and 750 nm (non-acidified readings are denoted by a subscript "o"). The stock concentrate was acidified in the cuvette using 2-4 drops of 3 N HCl. The absorbance after acidification was measured at 665 nm and 750 nm (acidified readings are denoted by a subscript "a"). Chlorophyll-a content was determined using the following equation (Strickland and Parsons 1972; Parsons et al. 1984): Chl-a (micrograms per liter)= [26.7*((ABS665o - ABS665a) - (ABS750o - ABS 750a))*1000]/l where:
Chl-a (micrograms per liter)= [26.7*((ABS665o - ABS665a) - (ABS750o - ABS 750a))*1000]/l
The stock solution was used to prepare six to ten standard dilutions of chlorophyll-a ranging from ~ 1.5 micrograms per liter to 500 micrograms per liter, plus a blank of 90% acetone. 90% acetone was used to dilute the standards. Fo and Fa were obtained for each standard by collecting initial fluorescence data on the Turner 10-AU fluorometer, and then acidifying the standard in the cuvette with 4 drops of 3 N HCl. The cuvette was briefly vortexed before determining Fa. The actual concentrations of the working standards were computed from the spectrophotometrically determined concentration of the stocks. A standard curve of chlorophyll-a concentration vs Fo-Fa was prepared. Each filter was placed into a 20 ml scintillation vial and extracted with 10 ml of 90% acetone. The extract was incubated for ~12 hours under cold (<0C), dark conditions. After incubation, the extract was briefly vortexed and 4 ml dispensed into the cuvette; the cuvette was inserted into the Turner 10-AU fluorometer. After Fo was determined, the sample was acidified with 4 drops of 3 N HCl, vortexed, and Fa was determined. The cuvette was rinsed three times with DI water and three times with 90% acetone to ensure there was no cross contamination of sample or acid between samples. The cuvette exterior was wiped with Kimwipes. The Fo-Fa was determined for each sample and chlorophyll-a concentration (microgram per liter) was calculated by comparison with the standard curve as below: Chlorophyll-a (micrograms per liter) = (((Fo-Fa) - y-intercept)/slope) * (ml extracted/ml filtered) where:
Chlorophyll-a (micrograms per liter) = (((Fo-Fa) - y-intercept)/slope) * (ml extracted/ml filtered)
Below is an outline of changes that have occurred to the above chlorophyll-a method over the time frame of the LTER dataset 9697 season The above method was followed with the exception that 90% acetone was replaced with a 1:1 solution of 90% acetone and DMSO. Shoaf and Lium (1976) showed that the absorption spectra of chlorophyll a was identical in 90% acetone and a 1:1 solution of 90% acetone and DMSO. 0506 season The method using a 1:1 solution of 90% acetone and DMSO (Shoaf and Lium, 1976) was followed with the exception that the acidification step was omitted for standards and samples run on the fluorometer. This method (Welschmeyer 1994) was employed to eliminate fluorescence from Chl-b interfering with fluorescence of Chl-a. Besides the lack of acidification, the following configurations were made to the fluorometer as described by Welschmeyer (1994):
0708 season Midway through the 0708 season a change was made in the Welschmeyer (1994) method where the 1:1 solution of 90% acetone:DMSO in the stock concentrate was replaced with 90% acetone. All other parameters of the method remained unchanged. FRX L1-L5, HOR L1-L5, ELB L1-L4, WLB L1-L4 and VAN L1 were analyzed before the change was made; all other samples were analyzed using the revised method. 0809 season In all steps of the method, the 1:1 solution of 90% acetone:DMSO was replaced with 90% acetone.
Citations Holm-Hansen, O., C. J. Lorenzen, R. W. Holmes, and J. D. H. Strickland. 1965. Fluorometric determination of chlorophyll. J. Cons. Int. Explor. Mer. 30: 3-15. Shoaf, W.T. , Lium, B.W. Improved extraction of chlorophyll a and b from algae using dimethyl sulfoxide, 1976. Limnol. Oceanogr. vol. 21. pp 926-928
Strickland, J.D.H. and Parsons. 1977 (1972 ?). Spectrophotometric Determination of Chlorophylls and total Carotenoids. A Practical Handbook of Seawater Analysis Fisheries research Board of Canada, Bulletin. vol. Chapter IV.3. pp185-196 Welschmeyer. Fluorometric Analysis of Chlorophyll a in the Presence of Chlorophyll b and Phaeopigments. 1994. Limnol. Oceanogr. vol. 39. pp1985-1992