ESR 324 Environmental Systems II Lab 3 Winter 2007

Transcription

1 ESR 324 Environmental Systems II Lab 3 Winter 2007 Nutrient Distribution in a Forested Watershed Where: TA: Instructor: TBA, Portland State University, and field sites in the Tualatin Mts Kate Norton (knorton@pdx.edu) Alan Yeakley ( ; yeakley@pdx.edu) Note: Some methods in this lab use hazardous chemicals. Become aware of the location of the nearest first aid equipment and safety shower to the lab work area before conducting the lab. You are required to wear eye protection while conducting the wet chemistry portion of the lab, and are recommended to wear rubber/plastic gloves while handling chemicals. No food or beverages are allowed in the lab area. Note: As with other labs in the course, Fieldbooks will be collected at the end of each laboratory, including both field and chemistry lab sessions and evaluated. The data from all students will be collected and collated and redistributed via . The fieldbooks will be returned to you the following week. Every student is responsible for filling out a fieldbook for all the data collected from his/her group; groups should work to insure that all data are consistent from each group. Introduction Having completed analyses of the topographic, vegetation and soil characteristics of the same watershed in Forest Park in previous labs, we will now estimate concentration of phosphorus (P), an important nutrient, in each of four forest ecosystem compartments: canopy (live foliage); litter (dead foliage on the forest floor); upper soil layers; and streamwater. We hypothesize that (1) P concentrations will vary among these four ecosystem components and (2) that there will be a difference between cove and ridge sites in terms of P concentrations in the soil, foliage and litter. Such a simple 4 compartment depiction of a forest ecosystem ignores many factors, such as nutrient concentrations in woody biomass (stems, branches, twigs), in the shrub and herbaceous layers, and in deeper soil layers. This approach also ignores seasonal variation; for example, we will not obtain values for deciduous canopy at this time of year. Further, we are only getting a rough approximation of spatial variability and are not measuring phosphorus input in the precipitation or from mineral weathering. Regardless, sampling from the four compartments described above will give us a snapshot of primary compartments for this important nutrient, and will also give us a very preliminary view how phosphorus is distributed in the forest ecosystem. Methods

2 During the first three sessions of this lab, we will collect and prepare samples for analysis for the following ecosystem compartments: Live conifer foliage Conifer litter Deciduous litter O horizon soil A horizon soil Streamwater in first order tributaries Streamwater at second order outlet Biomass samples will be categorized by tree species, and biomass and soil samples will further be categorized by location (cove vs. ridge). There must be a minimum of two replicates in any category to conduct statistical testing. First Lab Session This objective of this session is to obtain canopy and litter samples for nutrient analysis. Based on the tree species encountered both in Lab 2, we will attempt to obtain representative live foliage, or canopy, and dead foliage, or litter, from each species. To get more representative coverage of the watershed as a whole, we will take samples both in high elevation sites, and in lower elevation sites. Further, we will segregate our samples by habitat type: cove, ridge. The class will be divided into teams. Each team will be assigned a habitat type for sampling. From each habitat type, collect canopy and litter samples for each species encountered, ensuring at least 10 specimens of each (at this time, only litter will be available from deciduous species). For deciduous litter, a specimen is a whole leaf; for coniferous canopy or litter, a specimen is a twig of needles at least 2 long. For coniferous litter, try to collect intact specimens if possible. Select specimens from different branches or ground locations. Complete the sample collection for one species entirely before going on to the next species (this minimizes cross-contamination and allows you to seal the sample bag as soon as possible). Second Lab Session The objective of this session is to collect soil and streamwater samples for nutrient analysis. Based on the soil survey information from Lab 2, we will attempt to collect soil samples from at least two of the major soil series identified in the watershed: Goble silt loam (high elevation samples) and Wauld very gravelly silt loam (low elevation samples). In each series, we will sample the O and A horizons, obtaining at least four replicates for each horizon (4 replicates x 2 horizons x 2 series x 2 elevations = 32 samples total). Also, we will sample streamwater from all three headwater streams at mid elevation, as well as the 2nd order stream at the watershed outlet culvert near Hwy 30. For each stream sampled, we will obtain three replicates, for a total of 12 samples. You will be divided into groups, conducting either soil or stream sampling at each elevation. Soil sampling will be conducted using a soil probe, if available. Collect at

3 least 50 g per sample, which means that multiple extraction sites will be necessary per sample bag. Mark each bag by horizon and series. Streamwater will be grab sampled, using DI rinsed 500 ml sample bottles. Before sampling, rinse out each bottle three times with streamwater. After rinsing, fill each bottle completely allowing as little head space (i.e., air remaining in the bottle) as possible. Third Lab Session In this session, you will further prepare soil and vegetation samples for wet chemistry analysis. Soil samples Prior to this session, the TA prepared soil samples by drying at oven temperature (105 C) for 24 hours. For oven dried soil samples, a subset of the class will sieve each soil through 1.7 or 2.0 mm mesh (#12 or #10 sieve) to remove rocks and biotic matter such as roots. Extraction of phosphorus from soil samples will be done using Bray and Kurtz No. 1 method (Bray and Kurtz, 1945): 1. Weigh 2.5 g of 2.0 mm (or 1.7 mm) dry-soil into a 50 ml polypropylene test tube with cap. 2. Add 25 ml of extracting solution (mixture of 0.03M NH 4 F and M HCl). 3. Cap the test tube tightly and immediately shake suspension for exactly 1 minute by hand. 4. Immediately filter through Whatman GF/C filter paper using vacuums filtration into clean 50 ml polypropylene test tube. The filtration procedure should not exceed 10 minutes. Clearly label all test tubes. All persons conducting shaking and filtering will wear safety goggles. Vegetation samples The remainder of the class will prepare leaf samples, both from the canopy (live) and litter (dead). Combine same species samples taken from same location into one set of sample (e.g., all litter ALRU from upper ridge into one, all live PSME from lower cove into one). You will separate needles from the twigs and then grind the needles into a fine powder using mortar and pestle. Between each sample grinding, you will rinse the mortar and pestle with a dilute acid and then distilled water to prevent crosscontamination. Place ground samples into clean labeled ziplock bags. All persons conducting rinsing will wear safety goggles.

4 Intermediate Preparations The following steps will be performed by the TA prior to the Fourth Lab Session. Even though you will not perform these steps, you should include these steps in your lab report. All ground vegetation samples will be ashed prior to phosphorus extraction by TA. Method for dry ashing and extraction of phosphorus will follow Kalra and Maynard (1991). Dry ashing (ignition) for P 1. Place 0.50 g of ground plant material into acid washed and oven dried crucibles. 2. Place crucibles in a muffle furnace and ash the samples at 470 ±5 C for 16 hrs. 3. Moisten ash in crucible with 8-10 drops of water followed by 3 ml of 5 M HCl. 4. Place crucibles on hot plate (at low temperature 80 C) and add 0.25 ml of concentrated HNO 3. Evaporate to dryness to solubilize phosphate and precipitate silica. 5. Moisten dried salts from step 4 with 3 ml % M HCl and warm on hot plate. Add 5 ml water and maintain heat to dissolve salts. 6. Transfer solution while hot with distilled water to 50 ml volumetric flask through Whatman 42 filter paper. TQ to 50 ml with distill water and save aliquot for analysis. Streamwater samples will be analyzed by the TA for Soluble Reactive Phosphorus (SRP). Concentrations of SRP will be determined using the Ascorbic Acid Method (Wetzel and Likens 1991). SRP by Ascorbic Acid Method Preparation of phosphorus standard solution: Add oven dried g KH 2 PO 4 to a volumetric flask with 1 ml of chloroform and dilute to 1 liter with distill water. This makes 50 mg P/l stock solution. Series dilution of this stock solution can produce desirable concentrations for constructing a standard curve. Colormetric determination of phosphorus concentrations: 1. Filter streamwater sample through Whatman GF/C filter paper. 2. Pipette 25 ml of filtered samples and standards into 50 ml test tubes with lids. Add 2.5 ml of composite reagent (100 ml of 2.5M H 2 SO 4, 20 ml of 0.136%(w/v) potassium antimonyl-tartrate, 40 ml of 3% (w/v) ammonium molybdate and 40 ml of 5.4% (w/v) ascorbic acid. Combined in that order to make up 200 ml), cap and shake well.

5 3. Incubate for >10 min. but < 2 hrs. Read samples using a 5 cm path length cell at wavelength of 880 nm. Fourth Lab Session Colorimetric Determination of P We will use Vanadomolybdophosphoric yellow color methods (Kalra and Maynard, 1991) to measure amount of phosphorus in soil and vegetation samples. All methods are colorimetric based, meaning that the intensity of the color change is compared to standards with known concentrations to approximate the concentration of nutrients in the sample. Vanadomolybdate reagent preparation (prior to lab by TA). Dissolve 25g ammonium molybdate in 400 ml water (solution A). Dissolve 1.25 g ammonium (meta) vanadate in 300 ml of boiling water. Cool, add 250 ml concentrated HNO 3 and cool (Solution B). Add solution A to solution B and make up to 1L in a volumetric flask. Standard solution preparation (prior to lab by TA). Measure 0, 1.0, 2.5, 5.0, 7.5, 10 and 20.0 ml of 50 mg/l P stock solution in 50 ml volumetric flasks and follow steps 4 to 7 described below. Obtain concentrations of 0, 1.0, 2.5, 7.5, 10.0, and 20.0 mg/l P (in 50 ml volume), respectively. Vanadomolybdophosphoric yellow color method 1. Turn on Spec 20D and follow instruction written on the spectrometer to calibrate at 470nm. 2. First you need to construct a standard curve for spectrometer you are working with. Obtain standards solutions ( 0, 1, 2.5, 5, 7.5, 10 and 20 mg P/l) from your TA, fill up test tubes 2/3 and read the standards at 470nm. Make sure to record concentrations of standard and measured % transmittance in your lab notebook. 3. Pipet 10 ml aliquot of the plant digest or soil extracts in a 50 ml volumetric flask. Dilute to about 30 ml with distill water. 4. Add four drops of 2,4-dinitrophenol indicator. 5. Add NH 4 OH (about 5M) dropwise until yellow color just appears. This part is very difficult to observe because your sample solution is yellow to begin with. Look for darker yellow color change. 6. Add 10 ml vanadomolybdate reagent. 7. Make up to volume (i.e., 50 ml) and mix thoroughly. Use Parafile to cover opening while shaking. 8. Read P concentration at 470 nm after >10 min. but <2hrs. Make sure to rinse your cells (test tube used for spectrophotometers) with distill water in between readings. Keep your cells free of finger prints. Use Kimwipes to clean cells (do not use paper towel).

6 Construction of a standard curve and calculations of P in soil and vegetation 1. Construct a standard curve using a graph paper (transmittance in Y-axis and P concentration (mg P/L) in X-axis). 2. Using the standard curve you just constructed, find P concentrations (mg/l) in 50 ml solution based on measured % transmittance for each sample. 3. Use following equation to calculate P (mg/kg ) in soil or vegetation sample. ( / kg) P mg vol of digest ( ml) 50 = P ( mg / L) in50 ml solution weight of sample( g) vol of digest used to develop color ( ml) For soil samples: volume of digest is 25 ml, weight of sample is 2.5 g, and volume of digest used to develop color is 10 ml. For vegetation samples: volume of digest is 50 ml, weight of sample is 0.5 g, and volume of digest used to develop color is 10 ml. All analysis and calculations must be completed during the laboratory period. Each group is responsible for reporting their results on black board (there will be a summary table for everyone to share data). Before you leave the lab, make sure to copy all necessary data for your final lab report. Results Use t-tests and ANOVA tests to analyze the hypotheses of the lab. Present your results both in terms of the specifics of the statistical tests used and graphically. Discussion Discuss the significance of the results of the hypotheses tested in this lab. Include discussion of how extensive these four compartments are for the watershed. Discuss compartments that are being ignored in our sampling, and speculate on how much of an error it might cause to ignore them. Discuss how seasonal changes (particularly with respect to solar radiation and hydrology) would cause phosphorus to move, either among compartments in the watershed or out of the watershed entirely. Consider any field observations you have made that may be relevant to understanding nutrient locations in the foliage, litter, soil and streams. Include any references from previous studies that you might find in the literature (for example, Sollins et al. 1980) that might support the conclusions you draw.

7 References Bray, R.H., and Kurtz, L. T Determination of total, organic, and available forms of phosphorus in soils. Soil Science. 59: Kalra, Y.P., and Maynard, D.G Methods manual for forest soil and plant analysis. For. Can., Northwest Reg., North. For. Cent., Edmonton, Alberta. Inf. Rep. NOR-X-319. Sollins, P. et al The internal element cycles of an old-growth Douglas-fir ecosystem in Western Oregon. Ecological Monographs 50: Wetzel, R.G., and Likens, G.E Limnological Analyses, 2nd. ed. Springer-Verlag, N.Y. 391 pp.