Benthic algae chlorophyll measurements for Rowley River tidal creeks associated with long term fertilization experiments, Rowley and Ipswich, MA.

Benthic Algae (Chlorophyll a) Sampling Protocol for TIDE FIELD Frequency: Monthly, May thru September Field Equipment (stored in the TIDE benthic algae tool box): 50 ml syringes with tips cut off, knives, rulers, labeled 50 ml centrifuge tubes, extra labels and centrifuge tubes, pencil At each plant transect collect the following number of samples in each habitat: 3 mudflat (MF), 3 filamentous algae (FA), 3 tall creekbank Spartina alterniflora (TSA), 3 Spartina patens (SP), 3 short Spartina alterniflora (SSA). Habitat: Collect samples at low tide in order to access mudflat, filamentous algae, tall creekbank S. alterniflora and S. patens. , FA, and TSA habitat types. Use transect as guideline for picking sample locations, but do not work directly along the transect line. Choose general sampling location and toss syringe to “haphazardly” pick exact spot. To collect sample: slowly push down syringe while pulling up on the plunger (try not to compress the sample). Try to get as far down as possible and then slowly pull the tube out of the substrate. Pull the plunger out and stick it into the opposite end of the syringe. Using ruler to measure, push out 2 cm of sample into the appropriately labeled centrifuge tube. (Be careful to measure two centimeters as carefully as possible each time. Calculations of chlorophyll after analysis assume equal volumes of sediment in each sample.) MF: Take sample in exposed horizontal mud flats along creek channel during low tide. Avoid inserting syringe into an area with root masses and fibers, it is generally very easy to sample in the mud flats. FA: Take sample along creek wall below TSA roots layer in the obvious filamentous algal zone. TSA: Same as mudflats. SP & SSA: Sampling is more difficult, because of the dense roots. Cut around cylinder with knife as you are inserting syringe into ground. Once syringe is inserted far enough cut a cone shape underneath syringe with knife and pull out syringe at an angle in order not to lose the sample. Collection takes about a half day per creek with two people. Once samples are collected, wipe off as much mud on the outside of the tubes and store in labeled plastics bags. Samples must remain frozen until analysis. LAB EXTRACTION Step 1: Extraction (typically done in the pm the day before analysis on spectrophotometer) Supplies: 50-ml polypropylene centrifuge tubes containing sample (frozen and in the dark) 100% acetone 90% acetone Coolers with ice Re-pippeter Latex (non-nitrile) gloves Considerations: Chlorophyll degrades easily. As much as possible, keep samples cool and dark. Work in a hood to minimize your exposure to acetone fumes and cap samples tightly when not in use. Acetone eats many plastics so be mindful of the materials you are using to store and transfer samples. Acetone will also wash away sharpie- so keep track of sample numbers and protect your labels. Avoid using acid-washed equipment to minimize potential acid-contamination of samples. Length of extraction should be around 16 hours, so plan to time your sample prep and analysis accordingly. Prep only the amount of samples you can feasibly analyze in one sitting. Usually no more than 40- 50 samples at a time. Prep samples with acetone: 1. Allow samples to thaw at room temperature for about two hours before putting in coolers. Samples should be thawed, but cool to the touch. 2. Clean any excess mud off of sample containers and transfer thawed samples to coolers with ice 3. Store acetone solution in coolers on ice 4. Under hood, use a re-pippeter to add 25mls of 100% acetone to each tube 5. Shake each sample vigerously by hand to break up sediment at the bottom of each tube 6. Place tubes back on ice in cooler. Samples maybe sonicated or shock chilled to further break algal cells. 7. Centrifuge sample prior to reading on the spectophotometer. Reference creeks in all years are West and Nelson Nutrient Enriched creeks are Sweeney and Clubhead. Sweeney was fertilized every summer from 2004-2016. Clubhead was fertilized in 2005 and then from 2009-2016. All major creeks have two branches determined by direction at confluence while facing upstream (Left or Right). Plant transects run perpendicular to creeks at various points with a front pole at the creek bank and a back pole an the high marsh. Transects are in the order of 1, 2, 4, 3 or blue, green, red, yellow from confluence of branch. MF= Mud Flat TSA= Tall Spartina alterniflora SP= Spartina patens SSA=Short Spartina alterniflora FA= Filamentous algae