Six to eight sites in the Plum Island Sound estuary , the mainstem of the Parker River and Rowley River, in areas close to where other clam samples were being taken for other studies, were selected for monthly chlorophyll sampling to determine the concentration of phytoplankton at these sites. Roughly once a month, two 1L brown Nalgene bottles of water were sampled adjacent to each site from a boat at high tide. The water was kept cool and dark during transport to the lab, and filtered within three hours of collection. The procedure for filtration is outlined below: 1. Using square-ended forceps (to prevent poking a hole in the filter), place a single Whatman GF/F filter (2.5cm) onto a Gelman filter funnel. Make sure the funnel forms a tight seal with the base.2. The filter funnel should be attached to a vacuum system. We use a home-made PVC manifold that holds several funnels, and each funnel has a valve to open/close the vacuum. The manifold is attached to heavy-walled tygon tubing and a large vacuum flask (5 gallon glass water bottle). A second hose runs from the carboy to the vacuum pump. Use a moderate vacuum for the filtrations (ca. 100 to 200 mm Hg, or <7 in. Hg vac).3. Open the vacuum and pour in a pre-measured amount (using a clean graduated cylinder) of sample water. This is the tricky part. You may have to use trial and error to determine how much water you can filter before the filter clogs. Samples closer to the mouth of Plum Island Sound will require larger volumes (500-1000ml) than samples up the Parker River (150-700ml) depending upon season and discharge. Filter water samples in duplicate, be sure to record the total volume of water filtered for each duplicate sample.4. Using square-tipped forceps, gently fold the filter in half, with the side containing the sample on the inside of the fold. Remove the filter, keeping it folded, and place the filter between a small piece of aluminum foil 5. Label the outside of the aluminum foil (a square piece with dimensions of 5 x 5 cm) with the sample identification number. Use a black sharpie pen and make sure the ink dries (sharpie ink will destroy the pigment analysis….and the filters are magnets for sharpie ink!)6. Bend the edges of the foil to make sure the filter is sealed within the foil. Place the foil in a freezer (the colder the better). Keep the sample completely frozen and in the dark until you are ready to conduct the analysis To extract chlorophyll from the filters, the method outlined by the US EPA was followed: EPA Method 445.0, In Vitro Determination of Chlorophyll a and Pheophytin a in Marine and Freshwater Algae by FluorescenceElizabeth J. Arar and Gary B. Collins, Revision 1.2, September 1997 In short, the samples were placed in a test tube with 90% acetone and vortexed. They were kept cold overnight to allow time for the chlorophyll to be extracted, then vortexed again and analyzed on a Turner Trilogy Fluorometer. The fluorometer was calibrated with a chlorophyll standard curve using chlorophyll standards purchased from Turner Designs (standards had known concentrations and were then diluted to get a standard curve of 5 concentrations of chlorophyll ranging from low (0) to high (approx 200ug/L). After getting a chlorophyll reading, samples were acidified using 0.1NHCL to get the phaeophytin-a concentrations. Formulas for calculating chlorophyll-a and phaeophytin-a can be found in the Turner Trilogy Fluorometer user manual, page 36.
