PARTNERSHIP FOR THE DELAWARE ESTUARY Science Group. Prepared By: Danielle Kreeger

Transcription

1 PARTNERSHIP FOR THE DELAWARE ESTUARY Science Group Collection of Seston for Particle Counting, Weight-On-Ignition Analysis or Proximate Biochemical Analysis Date Prepared: 6/1999 Date Revised: 8/2006 (v2 by DK) Date Revised: 6/2012 (v3 by DK) Date Revised: 8/2017 (v4 by DK) Prepared By: Danielle Kreeger Suggested Citation: Kreeger, D Collection of Seston for Particle Counting, Weight-On- Ignition Analysis or Proximate Biochemical Analysis. Partnership for the Delaware Estuary. PDE Method No pp. PDE-Method (8/2017) Page 1

2 Collection of Seston for Particle Counting, Weight-On-Ignition Analysis or Proximate Biochemical Analysis Partnership for the Delaware Estuary (PDE) Method Danielle Kreeger Description This method describes the collection of seston from ambient water. Seston is defined as microparticulate material too small to be seen my the human eye. Although subtle differences can found in the exact particle size specifications comprising seston, here we consider this to included particles that are large enough to be retained on a glass fiber filter having an approximate retention of 0.7 µm (particle diameter) and small enough to pass through a 100 µm sieve. The purpose of this method is to collect seston from natural water samples for laboratory analysis of concentrations and relative percentage composition of total suspended solids (a.k.a. total particulate material), particulate organic content (POM), proximate biochemical composition (protein, lipid and carbohydrate contents), and sometimes also for other metrics such as elemental composition (C, N, P) and stable isotope composition. A method is also described for collection and fixing of non-filtered, but sieved water for analysis of seston particle concentrations and sizes. These procedures are typically used by our research team in studies of the food quantity and quality for suspension-feeding animals (bivalves, zooplankton); however, their utility also extends to general studies of ambient total suspended material. Summary of Approach Seston is collected from ambient water bodies that include fresh, brackish and saltwater systems, depending on the nature of the study. Typically, water is collected just below the surface into either 4 L cubitainers, or 10 or 20 L carboys. Replication per study site should be 3-4 field samples per sampling station/time. Water samples should be kept at ambient or cooler temperatures and shaded until processing in the laboratory within 24 hours. This procedure is also suitable for the analysis of cultured phytoplankton or any other organic microparticulate material. The first step in analysis is to sieve water to 100 µm with a Nitex screen sieve (note: some studies use 53 µm sieve), which is to eliminate larger debris and particles that is not efficiently captured as food by suspension-feeders. After sieving, subsamples of each water jug are used for the various analyses described. For analysis of particle concentrations using a Coulter Counter/Multi-sizer or hemacytometer, a 20 ml sample is fixed. For analysis of weights and proximate biochemistry, one replicate seston subsample is collected for each desired metric, by filtering onto prepared glass fiber filters using vacuum filtration. Filters will be then be frozen until laboratory analysis. Typically, for standard PDE-Method (8/2017) Page 2

3 analysis of proximate biochemistry and weight-on-ignition, four replicate subsample filtrations are needed per jug of water, giving one filter each for protein, lipid, carbohydrate and ash analyses. Hence, if only triplicate samples are sought per station/time, then 12 filters need to be collected for that station/time (4 per bottle/jug); if four replicates are sought, then 16 filters are needed. Where resources permit, 4 replicate bottles/jugs are recommended so that there is a greater likelihood of having at least 3 replicates for statistics, should some problem occur with any of the analyses. If additional seston analyses are included (e.g., for stable isotopes, particulate nutrients/stoichiometry, then additional filtered replicates should be collected. Typically, for weight, biochemistry, and particle concentrations, 4 L per sample is sufficient except when waters are oligotrophic, in which case larger volumes will be needed. Equipment Cubitainers or carboys (4L to 20L), 1 per sample replicate Coolers for holding water samples; add ice if air temperatures are warmer than water Sieve, 53 or 100µm Funnel (optional) 20 ml blood cell counter vials (e.g. Coulter), 1 per sample plus extras for blanks Acid-Lugols (4 drops per 20 ml particle sample, request DK-SOP-31) Filter forceps (e.g. Millipore type that do not puncture filters) Glass fiber filters having average retention of 0.7 µm (47 mm diameter preferred, but 25 mm diameter are ok). Whatman type GF/F is the standard, although VWR and other suppliers sell equivalent versions Plastic filter storage holders (special ones such Anyalslides pr Petrislides can be purchased and are preferred since they require less freezer space, but small plastic Petri dishes work as well) Aluminum foil Vacuum filtration system. Where possible, a multi-channel setup speeds sample filtration. Do not exceed 25 psi. Graduated cylinders and adjustable pipettes for volume measurement, tips (for seawater only) 0.5 M ammonium formate (5 ml per filter subsample) (for freshwater) distilled water (5 ml per filter subsample) Drying oven set at 60 o C Muffle furnace capable of heating to at least 450 o C Freezer for holding seston filters once processing is complete Procedures (not needed for particle concentration analysis) Use only Millipore type forceps to handle filters. Never touch them Pre-ashing. All glass fiber filters must be pre-combusted to ensure that no organics contaminate the filters. A sufficient number of filters for the sampling is removed from the box and arranged in groups of ten on an aluminum foil sheet. Each group of 10 filters is then arranged on a separate sheet of foil, which is folded overtop the filters, keeping the shiny side PDE-Method (8/2017) Page 3

4 outward away from the filters. Aluminum foil packets are then be pre-combusted at 450 C for at least 24 hours (or 500 C for at least 4 hours). Pre-ash enough filters for all replicates needed Pre-weighing. For each bottle/jog of water, one filter replicate is used for weight-onignition (a.k.a. loss-on-ignition ), and the remaining three filter replicates will be used for biochemistry. The replicate to be used for weight-on-ignition needs to be pre-weighed after it has been pre-ashed (step 23.1). Weights are determined on an analytical balance. The precision of the weight-on-ignition technique will depend on the carefulness of weighing on the analytical balance. Since only 1 mg or less of sample might be collected, the balance should be set at 5 places (± 10 μg). Balance should be routinely calibrated with a 10 mg standard weight. Place an inverted plastic weigh boat on the scale so that filters can be easily picked up. Tare balance. Typically, a 47mm diameter glass fiber filter will collect 5-25 mg of seston. The same balance should be used before and after filtering seston for weight-on-ignition. Weights should be measured only on desiccated samples, which can be accomplished by adding filters to be weighed into filter holder dishes, which are left to stand in a desiccator overnight prior to weighing. After weighing, each individual filter is added to a labeled filter holder (e.g. Petrislide) having a unique filter ID# Glassware preparation. All vacuum filtration glassware should be pre-cleaned to ensure it is as particle-free as possible. This is particularly important for the funnels and filter holders. A separate method (request DK-SOP-1) describes suitable glassware washing. A pre-rinse with 10 ml of 10 % HCl per funnel is suitable. Filter a few mls, let stand for 2 min, filter remaining acid, and then rinse with 10 ml particle-free distilled water (if distilled water has particles, it can be pre-filtered as well). Procedures Methods for filtering seston for weight and proximate biochemistry are described in step Methods for assessing particle concentrations, sizes and volumes are described in step Enter all notes, filter weights, and volumes filtered into lab notebook Sieve Water Sample. Regardless of the analysis below, all water samples must be shaken to mix and then passed through either a 53 µm or a 100 µm sieve prior to dividing into subsamples for various analyses. For this, a useful protocol is to set a funnel in a clean (prerinsed) 4 L cubitainer (or large beaker/pitcher) and the sieve is set in the funnel. Once sieved, the water sample should be mixed by inverting or plunging a graduated cylinder bottom, prior to each subsample for various analyses Seston Filtration for Weight and Biochemistry (and other metrics). All glass fiber filters are pre-combusted, and filters for weight analysis are additionally pre-weighed, as described in steps 23.1 and Use only Millipore style forceps to handle GFF filters. An example of a seston filtration datasheet is given in Appendix A. A sketch of the vacuum filtration apparatus set-up is given in Appendix B. This shows a 6-channel manifold, but single flasks and 3-channel systems are set up the same way. PDE-Method (8/2017) Page 4

5 Filter seston (or algae) by passing a known volume of mixed water. The desired volume of water should be as much as possible without clogging the filter. Typically, analytical accuracy is maximized with greater filtered seston weights. However, filter clogging could lead to problems with pore size bias and will take longer. Therefore, for each water type, a useful practice is to waste a filter first by running enough water through to cause clogging, defined as a marked slow-down in the pass-through rate. The volume that leads to clogging is then recorded, and subsequent volumes are set to be 90% of the clogging volume. The volume required depends on the density of particles. For seston, the volume required to clog a filter typically ranges between ml. For dense lab cultures of algae, the ideal volume will range from 25 to 100 ml generally. Vacuum pressure should be no greater than 20 psi to prevent filter breakdown, and 15 psi is preferred. Record volumes filtered as accurately as possible (i.e. if small volumes are filtered, deliver with a calibrated pipet) Rinse each filter and its funnel as soon as the water sample passes using 5 ml per funnel/sample of either distilled water for freshwater samples or 0.5 M ammonium formate for brackish or seawater samples. This rinse is mainly needed to wash any particles that cling to the funnel sides onto the filter. A 5 ml pipette is useful as a jet spray, ringing the funnel around with this volume. Seawater can leave salts behind, which could also bias the inorganic content of filtered particles if left on the glass, and the ammonium formate is an effective salt rinse Transfer each filter to a pre-labeled storage dish (e.g. Petrislide). Weight filters should be returned to their holders. Be sure filters are always facing up so the sestonladen surface does not contact the lid. Always handle filters by the edges where no seston is trapped. Ensure that filters do not tear while lifting from the filtration screens. If using a filter manifold, it is useful to first break the vacuum before attempting to remove the filter Seston Collection and Analysis for Particle Concentrations. Procedures are described for counting particle concentrations using either a hemacytometer or Coulter Counter. Hemacytometers are relatively simple to use, do not need calibration and will need to be used when a Coulter Counter is unavailable (e.g., for quick counts away from a Coulter Counter) or when interspecific counts are needed for mixed assemblages of particles. Coulter Counters are more rapid, accurate and precise than hemacytometers (if calibrated and used properly), and when used with a Channelyzer or Multi-sizer, they can provide much more information (e.g., volumetric concentrations, size-specific concentrations). One purpose of counting particle concentrations will be to calculate how much algae to deliver to suspension-feeding animals in feeding experiments. Since the size of algal cells can vary dramatically under different culture conditions, it is important to calculate algal rations on the basis of their volumetric concentration and not cellular concentration - cell volume has a closer relationship with cell ash-free dry weight (AFDW). A goal should be to pre-establish PDE-Method (8/2017) Page 5

6 volume:afdw regression equations for each algal species and culture condition prior to use in dosing rations in feeding studies. For characterizing the particle size distribution of natural seston with the Coulter Counter, 20 ml of water should be sampled and analyzed as soon as possible (within 24 hr), unless fixed with acid-lugol s (and then analyzed within 2 weeks). If the interval between collection and analysis is more than 4 hours, samples should be kept in the dark (wrapped with foil if needed) and in a cooler with ice or ice packs. Samples should never be frozen Hemacytometer Procedure. Wash the hemacytometer and cover slip with soapy water (e.g., 7X), rinse three times with tap water, three times with distilled water and dry with a Kimwipe; place cover slide on hemacytometer. Mix the algal or natural seston sample by shaking. Use Pasteur pipet to transfer a drop of the mixed suspension to the edge of the cover slide of the hemacytometer, and capillary action will pull the drop into the counting chamber. Repeat for the other side of the hemacytometer, but take care not to overfill. Count the number of cells on 8 squares of each side (try always to count the same 8 squares). The number of cells in 16 squares x 104 equals the cell concentration per ml. Repeat particle count in step 2.2 three times Coulter Counter Procedure. The Coulter Counter contains a vacuum pump attached to a narrow glass tube with a mercury manometer. When a sample is placed under the aperture nozzle, and a stopcock is opened on the instrument panel, the vacuum pump pulls the mercury out of its equilibrium position and resets the LED particle counter. After the valve is closed, the mercury gradually recedes to its equilibrium position, and while doing so, it gradually pulls approximately 750 μl of sample through a small opening on the aperture nozzle. Electronic sensors attached to the mercury manometer start and stop the counting of particles so that exactly 500 μl of sample is counted. As the water sample is drawn through the small aperture, particles disrupt the electronic field around the aperture and these disruptions are translated into counts on the LED display. Since the Coulter Counter operates on a semi-continuous flow basis, it is important to check the inflow and outflow reservoirs before and after using the instrument. The inflow and outflow reservoir should be kept between 1/4 and 3/4 full. Calibrate the instrument for particle size at least monthly. This is easily done with a suspension of uniform, pre-defined size particles (e.g., monolatex beads). If possible, use a particle size that is similar to the size particle being routinely counted. On the setup screen of the Channelyzer, enter the diameter of this particle under the calibration sub-menu. Run the sample and view the particle size distribution on the Channelyzer graph. Position the left and right cursor to surround the peak of the bead distribution, and press the "calibrate" button on the front of the Channelyzer. The particle diameter (x-axis) corresponding with the peak abundance (y-axis) should now equal the nominal bead size supplied by the manufacturer. PDE-Method (8/2017) Page 6

7 Dilute each sample of algae by adding 1 ml algae to 15 ml Isotone and mix by shaking. Dilute each sample of natural seston by adding 10 ml of sample to 10 ml Isotone (at least 50% electrolyte is required for freshwater samples but saline samples can be analyzed without dilution) and mix by light shaking. Repeat twice to give 3 samples. Zoom in on the appropriate Channelyzer window. If the Multi-sizer is being used, prepare the software for data capture. Press the reset button on the front of the Channelyzer. Shake each sample to mix just prior to counting. Put the sample under the aperture (be sure the foil sensor is immersed) and open the upper stop-cock one quarter turn. You should see the mercury column move and the LED counter reset to zero. Close stopcock, and the counter will automatically count the sample for seconds. Count each replicate sample twice and record the particle concentration within the windowed range of the Channelyzer. Repeat a count if the two values differ by >10%. The count, which is the concentration of cells per 0.5 ml, should be between 500 and 20,000 for best accuracy - if it is not, prepare new samples for counting using more appropriate dilutions. Correct each raw count for coincidence using Appendix B, if needed. Note that some models of the Multisizer have newer software that automatically corrects for coincidence. In addition to the algal samples, also count the particle concentration in the water used to suspend and dilute the algae as a blank. Subtract the particle concentration of the blank from the coincidence-corrected sample concentration, multiply by 2 to obtain the sample count per ml, and correct for the dilution (e.g., multiply by X times) Volumetric Counts Using Coulter Multi-sizer. Although volumetric concentrations can be estimated using only the Channelyzer (tedious calculation of geometric mean volume for each of many channels), the Multi-sizer is much more accurate and fast. The software does nearly all of the work. Select the "edit listing" menu and check the boxes "differential" and "cumulative volume". Then select the function "listing" to view the cumulative volume data in column form, with each value corresponding to the total volume of particles with that particle size channel (256 channels measured per count). For seston samples, only the volume of particles between 2 and 63 μm may be needed. For algal culture, a particle size window should be pre-selected to quantify consistently so that any bacteria or other non-algal particulate material are not included in the estimate of volume. Therefore, for each measurement, only record the volume of particulate material within a designated range rather than the whole 256-channel range recorded by the Multi-sizer. The cumulative volume associated with each size channel can be viewed from the "listing" and recorded on a separate table (Appendix C). The cumulative volume associated with the lower and upper limits of each sample's pre-selected range should PDE-Method (8/2017) Page 7

8 be noted, and the difference between these values is equivalent to the total volume of particulate material within that size range. This particle volume should be divided by the volume of sample analyzed (i.e., each measurement counts 0.5 ml), and corrected for any dilution factor. The resulting "volumetric concentration" (VC) has the units volume per volume, usually μm3 per ml. The VC of a sample can then be divided by the corresponding concentration of ash-free dry tissue (units = mg per ml; from PDE- Method-07) to calculate the relationship between sample volume and AFDW (units = μm3 per mg). We will establish a regression equation for this volume:afdw relationship for each type of algae. PDE-Method (8/2017) Page 8

9 Appendix A Project: Location/Study Site: Date/Time Samples Collected in Field: Person(s) Sampling in Field: Wetlands Group - Seston Analysis Sample Sheet Pre-sieving? Date/Time Samples Processed in Lab: Person(s) Processing in Lab: Notes: Sample Type Replicate 1 Replicate 2 Replicate 3 Replicate 4 Sample Volume Sample Volume Sample Volume Sample Volume No. (ml) No. (ml) No. (ml) No. (ml) Weight Pre-weight # > Protein Lipid Carbohydrate Elemental C & N Chlorophyll Isotopes Particle Sizes (Multi-Sizer) PDE-Method (8/2017) Page 9

10 Appendix B PDE-Method (8/2017) Page 10

11 Appendix C Cell Number in AW Total Volume in AW (μm 3 ) Sample Initials Dilution Factor Analysis Window (AW) Replicate Raw (#/ 0.5 ml) Coincidence Correction (#/0.5 ml) Blank & Dilution Corr. (#/ml) Raw (μm 3 /0.5 ml) Blank & Dilution Corr. "Vol. []" (μm 3 /ml) Mean Cell Volume (μm 3 ) Estimated Concentration of AFDW (mg ml -1 ) PDE-Method (8/2017) Page 11