Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9
Objectives Students will be able to: · define alkalinity and hardness in water. · identify methods used to measure and analyze the alkalinity and hardness in water samples. · identify methods used to determine the amount of specific nutrients in water. · interpret data from nutrient standard calibration curves. · explain methods used to measure total suspended solids in water samples. · calculate the total suspended solids in water samples. · explain methods used to measure turbidity. · evaluate and compare turbidity data against specified standards. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 2
Objectives cont. Students will be able to: · describe procedures used for determining biochemical oxygen demand. · explain methods used to determine algal biomass and biovolume. · compare and contrast spectrophotometers and fluorometers. · identify methods used to measure algal chlorophyll. · estimate the biomass and biovolume for periphyton samples. · describe procedures used to measure bacterial colonies in water samples. · determine methods used to measure biomass of aquatic vegetation. · identify methods used to measure benthic invertebrates and zooplankton. · analyze the properties of benthic sediments. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 3
Basic water quality assessment – lab · Goals – lectures and labs focus on analyzing samples in lake surveys and on parameters used in lab experiments · Water chemistry – · alkalinity and hardness · nutrients by colorimetry and kits · suspended sediments (TSS) · turbidity · organic matter (BOD), color Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 4
Basic aquatic community assessment · Algae and bacteria (chlorophyll-a, microscopy, plate counts) · Aquatic vegetation and attached algae (periphyton) · Zooplankton · Sediment bulk properties · Benthic organisms · Microbial pathogen indicators · Fecal coliforms and E. coli Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 5
Alkalinity and hardness Photo of p. H test Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 6
Alkalinity and hardness - what is it? · Alkalinity: a measure of the ability of a water sample to neutralize strong acid · Expressed as mg Ca. CO 3 per liter or microequivalents · Alkalinities in natural waters usually range from 20 to 200 mg/L · Hardness: a measure of the total concentration of calcium and magnesium ions · Expressed as mg Ca. CO 3 per liter Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 7
Alkalinity and hardness - how to sample · Usually collected at the surface in lakes (0 to 1 m depth) · Keep the sample cool (4 o. C refrigerated) and out of direct sunlight Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 8
Alkalinity and hardness- why measure? · The alkalinity of natural waters is usually due to weak acid anions that can accept and neutralize protons (mostly bicarbonate and carbonate in natural waters). · Usually expressed in units of calcium carbonate (Ca. CO 3) · The ions, Ca and Mg, that constitute hardness are necessary for normal plant and animal growth and survival. · Hardness may affect the tolerance of fish to toxic metals. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 9
Alkalinity – analysis · p. H meter · Buret* · Thermometer · Magnetic stirrer and stir bar · Top loading balance Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 10
Alkalinity- analysis · Reagents · 0. 04 N H 2 SO 4 (see method for details on preparation) · Total alkalinity analysis involves titration until the sample reaches a certain p. H (known as an endpoint) · At the endpoint p. H, all the alkaline compounds in the sample are "used up" · The amount of acid used corresponds to the total alkalinity of the sample · The result is reported as milligrams per liter of calcium carbonate (mg/L Ca. CO 3) · The value may also be reported in milliequivalents by dividing by 50 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 11
Alkalinity- analysis or Where: B = m. L titrant first recorded p. H (i. e. , to p. H = 4. 5) C = total m. L titrant to reach p. H 0. 3 unit lower, and N = normality of acid (titrant) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 12
Hardness – analysis · Hardness is, ideally, determined by calculation from the separate determinations of calcium and magnesium. Hardness, in units of mg Ca. CO 3/L Where Ca and Mg are in mg/L Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 13
Alkalinity and hardness – analysis There also titration test kits available for both alkalinity and hardness www. lamotte. com www. hach. com Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 14
Nutrients Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 15
Nutrients: colorimetry & spectrophotometry · Overview of the colorimetric analysis of the nutrients nitrogen and phosphorus using spectrophotometry · Specific techniques for students to review in or out of class included: · developing calibration curves · QA/QC : standards, spikes, etc. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 16
Nutrients - how to sample · Usually collected from discrete depths · Keep samples cool and dark · Freeze unless you can run in <24 hrs · Follow APHA recommendations Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 17
Nutrients: sample processing · Total phosphorus (TP) and total nitrogen (TN) analyses are made with whole, or raw, water · Unfiltered sample · Dissolved (soluble) fractions are with a filtrate · Includes ortho-P, ammonium, nitrate and nitrite · EPA and most states require the use of a membrane filter with a nominal pore size of 0. 45 um · most researchers use glass fiber filters Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 18
Nutrients: colorimetry & spectrophotometry Principles: 1. Higher concentration of color = higher absorbance, as measured by a spectrophotometer · add a dye that binds specifically to nutrient of interest · measure the increase in “color” as an estimate of analyte concentration Developed by: Axler, Ruzycki 2. Prepare calibration standards - solutions with a range of nutrient concentrations 3. Compare sample absorbances to calibration standard absorbances to estimate sample concentrations Updated: Dec. 29, 2003 U 3 -m 9 a-s 19
Nutrients: colorimetry & spectrophotometry 4. Add reagents to develop color Low …. …. to ……. High Phosphate concentration Developed by: Axler, Ruzycki 5. Compare · using a chart or color wheel · using a colorimeter · determining the absorbance using a spectrophotometer Updated: Dec. 29, 2003 U 3 -m 9 a-s 20
Color comparators and colorimetry Test Kits – There are many brands available Color Tube Color Disc Pocket Colorimeter Images from www. hach. com Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 21
Color measuring instruments Hach DR 2400 portable spectrophotometer Developed by: Axler, Ruzycki • Bausch & Lomb spectrophotometer 20 Updated: Dec. 29, 2003 U 3 -m 9 a-s 22
Calibration standards Standards are made from a concentrated stock solution that is precisely diluted to create “working standards” that are used and then discarded Ortho-P: NH 4 -N and NO 3 -N: Use dried KH 2 PO 4, K 2 HPO 4, Use dried NH 4 NO 3 as a dual Na. H 2 PO 4 or Na 2 HPO 4 standard (50% of each form) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 23
Water chemistry “ 101” Procedure: · See specific analyses · Reagents are added to each sample and standard identically · Mix after each step · Incubate at room temp or in water bath for 20 min to ~ 2 hrs, depending on the analyte Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 24
Standard calibration curves NH 4 -N standards Developed by: Axler, Ruzycki Good straight line fit: ABS = a + b*[Conc] Updated: Dec. 29, 2003 U 3 -m 9 a-s 25
Estimating concentrations So, if sample #3 had an absorbance of 0. 290… Its concentration would be ~ 0. 33 ppm N … N Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 26
Standard curves – troubleshooting Example #1 – Live with it or re-run the batch #1 Errors in preparing the 0. 25 and 0. 50 ppm standards perhaps ? #2 Example #2 – Fit a straight line from 0 -1000 and a 2 nd line from 12002000 ug. N/L · Use non-linear quadratic instead of a line for 0 -2000 ug. N/L · The line becomes nonlinear after ABS ~ 1. 0 (~ 1000 ug. N/L) Re-read in smaller cuvette or dilute and re-run Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 27
Some data from northern Minnesota lakes Calibration curve ABS = (-0. 0010) + (0. 00254)* P R 2 = 0. 9997 n=12 = std Sample #1 = 11. 2 ug. P/L Sample #1 - Replicate = 12. 6 ug. P/L Sample #1 + 50 Spike = 59. 4 ug. P/L Conclusion: % RPD = 100* (1. 4)/ 11. 9 = 12% The data are valid % R = 100* (59. 4 -11. 9)/50 = 95% Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 28
Total suspended solids and turbidity Sediment plume off the south shore of Lake Superior Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 29
Total suspended solids and turbidity • TSS and turbidity are two common measures of the concentration of suspended particles. • Suspended materials influence: • Water transparency • Color • Overall health of the lake ecosystem • Nutrient and contaminant transport Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 30
Total suspended solids - sampling · TSS sampling in lakes involves collecting whole water samples · No special handing or preservation is required but samples should be kept cool until analysis · Recommended holding time is 7 days if kept at 4 o. C (but the sooner the better) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 31
Total suspended solids - method 1. Filter a known amount of water through a pre-washed, pre-dried (at 103 -105 o. C), preweighed (~ + 0. 5 mg) filter 2. Rinse, dry and reweigh to calculate TSS in mg/L (ppm) 3. Save filters for other analyses such as volatile suspended solids (VSS) that estimate organic matter Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 32
Total suspended solids - method What type of filter to use? Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 33
Total suspended solids · Some examples of filter types: · Membrane filters retain sub-micron particulates and organisms · Glass microfiber filters are made from 100% borosilicate glass · Polycarbonate - offers precise pore size but reduced flow www. whatman. com Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 34
Total suspended solids – method · There are many different set-ups · attach funnels by clamp, screw-on, or magnetic base · plasticware useful in the field multiple towers Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 35
Total suspended solids · Necessary TSS equipment Analytical balance Drying oven Filter and petri dish Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 36
Total suspended solids · Calculate TSS by using the equation below: TSS (mg/L) = ([A-B]*1000)/C where A = Final dried weight of the filter (in milligrams = mg) B = Initial weight of the filter (in milligrams = mg) C = Volume of water filtered (in Liters) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 37
How do turbidity and TSS relate? A general rule of thumb: 1 mg TSS/L ~ 1. 0 - 1. 5 NTU’s of turbidity BUT – Turbidity scattering depends on particle size so this is only a rough approximation Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 38
Turbidity - meters · Most use nephelometric optics and read in NTUs (nephelometric turbidity units) · Field turbidity measurements are made with: · Turbidimeters (for discrete samples) · Submersible turbidity sensors (Note: USGS currently considers this a qualitative method) · Laboratory instruments: · Turbidimeters (bench models) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 39
Turbidity Turbidimeters Nephelometric optics • nephelometric turbidity is estimated by using the scattering effect suspended particles have on light • detector is at 90 o from the light source http: //www. bradwoods. org/eagles/turbidity. htm Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 40
Turbidity – units and reporting · Nephelometric Turbidity Units (NTU) · Standards are formazin or other certified material · JTU’s are from an “older” technology in which a candle flame was viewed through a tube of water · 1 NTU = 1 JTU (Jackson Turbidity Unit) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 41
Turbidity – formazin standards · Example of a set of formazin standards Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 42
Turbidity - · Here is a range of NTUs using clay Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 43
Turbidity – meters and probes · Bench and portable instruments and kits vs. ·Submersible Turbidimeters YSI 6820 with unwiped turbidity YSI wiping turbidity Hydrolab Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 44
Turbidity - methods · Comparability of different methods: · With the proliferation of automated in situ turbidity sensors there is concern about the comparability of measurements taken using very different optical geometries, light sources and light sensors. · The US Geological Survey and US Environmental Protection Agency are currently (August 2002) developing testing procedures for a field comparison of a number of instruments produced by different manufacturers. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 45
Turbidity - calibration · Turbidity free water = zero (0 NTU) standard · USGS recommends filtering either sample water or deionized water through a 0. 2 um or smaller filter to remove particles · WOW uses deionized water that is degassed by sparging (bubbling) with helium, to minimize air bubbles that may give false turbidity readings Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 46
Turbidity - standards · Standards range depends on anticipated sample values · Lakes - typically 0 -20 NTU · Streams and wetlands - 0 -20, 0 -50 or 0 -100 NTU · 2 non-zero standards typically adequate (response is linear) · Types of standards · Formazin particles (either from a “recipe” or purchase a certified, concentrated stock solution usually 4000 NTU) · Other commercially available materials, e. g. , polystyrene Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 47
Turbidity – standards Source Concentrations Table of standards Hach Company Suggested holding times 2 to 20 NTU Prepare daily 20 to 40 NTU Prepare monthly Standard Methods All dilutions (APHA 1995) Prepare daily EPA Region 5 Prepare weekly All dilutions Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 48
Biochemical Oxygen Demand (BOD) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 49
BOD · BOD measures the amount of oxygen consumed by microorganisms as they decompose organic matter, as well as the chemical oxidation of inorganic matter · The BOD test measures the amount of oxygen consumed during a specified period of time (usually 5 days at 20 o C) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 50
BOD 5 · DO is measured initially and again after a 5 -day incubation at 20 o C · BOD is computed from the difference between initial and final DO · The rate of oxygen consumption is affected by a number of variables: · temperature · p. H · the presence of certain kinds of microorganisms · the type of organic and inorganic material in the water Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 51
BOD – sample collection · Sample collection · Grab samples in clean, sterile containers (usually only surface sampling) · If analysis is begun within 2 hours of collection, cold storage is unnecessary · If analysis will be delayed > 24 hrs, store at or below 4 o C · Warm chilled samples to 20 o C before analysis Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 52
BOD - analysis · Equipment needed: · Incubation bottles · Air incubator or water bath thermostatically controlled at 20 +/- 1 o C · DO meter and probe Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 53
BOD · Reagents: · Dilution water – provides nutrients necessary for microorganism growth · Seed – a population of microorganisms capable of oxidizing the organic matter in the sample · Commercially available or freeze-dried culture · A “conditioned” bacteria source (effluent from a biological treatment source such as a wastewater treatment plant). · Glucose-glutamic acid standard Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 54
BOD – QA/QC · Assure quality with: · Seed control – determine the BOD of the seeding source · Dilution water blank – used to check for quality of unseeded dilution water and incubation bottle cleanliness · Steps to Include: · Read and record temperature of incubator · Prepare replicate bottles for dilution water blanks and seed controls · Include at least one set of replicate samples per analysis Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 55
BOD - procedure · Blanks · Prepare dilution water, bring to 20 o C and aerate · Add sufficient seeding material to produce a DO uptake of 0. 05 to 0. 1 mg/L in 5 d (dilution water) · Samples · Add sample to bottle and dilute. · Dilutions should result in a residual DO of at least 1 mg/L and DO uptake of at least 2 mg/L after 5 day incubation Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 56
BOD – procedure · Steps in procedure: · Fill bottles with enough dilution water so the stopper displaces all of the air, leaving NO air bubbles · Read initial DO · Incubate for 5 days at 20 o C · Read final DO · Calculate BOD 5 correcting for the exact duration Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 57
BOD Calculations · When dilution water is not seeded: · When dilution water is seeded: Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 58
Phytoplankton/Algae – counting methods Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 59
Algae- counting methods · Wet mounts · Filter · Counting chambers · Utermohl · requires an inverted microscope (light from above) · Sedgewick rafter chamber · Hemocytometer Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 60
Algae – counting methods Microscopes capable of magnifications of 100 X to 1000 X Compound microscope Inverted microscope Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 Less expensive inverted microscope U 3 -m 9 a-s 61
Algae- taxonomy · Use an algal taxonomic key that shows species from your geographical area · Phytoplankton are continually being described and re-classified so it’s essential for a good taxonomist to keep current (not easy by any means) · It’s a good idea to take photographs of slides for cataloging Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 62
Algae – determining biomass · Algal biomass (standing crop): · A quantitative estimate of the total mass of living organisms within a given area or volume · Biovolume estimates: · Identification to genus and species level · Calculate cell volume by approximation to nearest geometrical shape · Count cells over a known area of the slide so cells per unit volume can be determined · Chlorophyll Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 63
Algae – determining biovolume · Taxonomic keys often include questions about size · Determining size is basically like using a ruler. · The standard ruler for a microscope is called an "ocular micrometer, " which is fitted into the eyepiece of your microscope Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 64
Algae – determining biovolume Some formulas to estimate biovolume from cell dimensions (Wetzel & Likens 2000) B A A B A Rod Sphere Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 Ellipsoid U 3 -m 9 a-s 65
Algae – chlorophyll determination Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 66
Algae – chlorophyll determination · Measuring chlorophyll-a concentration remains the most common method for estimating algal biomass · Chlorophyll-a concentration has also been shown generally, when comparing lakes, to relate to primary productivity (Wetzel 1983) · Can be used to assess the physiological health of algae by examining its degradation product, phaeophytin Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 67
Algae – chlorophyll basics · Algal biomass is most commonly estimated by chlorophyll-a. · Units are ug/L or mg/L (ppb and ppm) · Detection limit depends upon method used Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 68
Algae – chlorophyll methodology · Spectrophotometry and fluorometry, utilizing 90% acetone extraction, remain the most commonly used methods · Spectrophotometry is most widely used but fluorometry is more sensitive and may be used when low levels of chlorophyll are anticipated or when handling large volumes of water is logistically difficult Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 69
Algae – chlorophyll sampling · 0 to 2 m integrated samples are usually collected for chlorophyll analysis · Samples must be kept cool and out of direct sunlight until filtered · Freeze moist filters until analysis Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 70
Algae – chlorophyll instrumentation · Spectrophotometer: · Visible with 1 -2 nm bandwidth · Matched cuvettes, 1 -5 cm · Fluorometer: · Requires excitation and emission filters specifically for chlorophyll measurement Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 71
Algae – chlorophyll filtration Apparatus - extraction · Prewashed 47 mm glass fiber filters (GF/C, GF/F, AE, or equivalent) · Gelman polycarbonate filtration tower or equivalent · Vacuum pump (5 to 7. 5 psi) · Centrifuge (clinical) · DIW/acetone (90%) washed 15 m. L Corex centrifuge tubes with caps Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 72
Algae – chlorophyll filtration (cont. ) · Filter a known volume of water through a GF/C filter · Volume filtered depends upon algal density · Add a few drops of saturated Mg. CO 3 solution near the end · When all the water has been pulled through, fold the filter into quarters and wrap in foil Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 73
Algae – chlorophyll storage · Wrap the folded filter in a square of foil, label, then freeze · Record the volume filtered, date, site, depth, replicate # all with permanent marker · Store the filter in the freezer at < 20 o C · EPA holding time for a frozen chlorophyll filter is 2 weeks Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 74
Algae – chlorophyll extraction & analysis · Chlorophyll extraction: · Tear filter into several pieces · Place in a test tube · Add 10 m. Ls of 90% acetone · Extract overnight at 4 o. C · Chlorophyll analysis: · After 18 -24 hr extraction, centrifuge to settle filter debris · Read absorbance or fluorescence of the supernatant Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 75
Algae – chlorophyll measurement · Measure absorbance of a 90% acetone solution blank at 750 nm and at 664 nm to correct for primary pigment absorbance · Record sample absorbance at 750 nm and 664 nm · Estimate phaeophytin by acidifying the sample. Record the absorbance at 665 nm and again at 750 nm · Run working standard solutions of purified chlorophyll-a (Sigma Chemical Co. Anacystis nidulans by the procedure used for the blank) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 76
Algae – chlorophyll and phaeophytin · What is phaeophytin? · Degradation product of chlorophyll · Absorbance wavelength (665 nm) is very close to that of chlorophyll (664 nm) Developed by: Axler, Ruzycki acid H Updated: Dec. 29, 2003 U 3 -m 9 a-s 77
Algae –spectrophotometry calculations Where: b = before acidification a = after acidification E 664 b - [{Abs 664 b(sample)–Abs 664 b(blank)}-{Abs 750 b(sample)–Abs 750 b(blank)}] E 665 a - [{A 665 a(sample)-Abs 665 a(blank)}-{Abs 750 a(sample)-Abs 750 a(blank)}] Vext = Volume of 90% Acetone used in the extraction (m. L) Vsample = Volume of water filtered (L) L = Cuvette path length (cm) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 78
Algae – chlorophyll QA · Quality assurance · There are no commercial QA check standards · Lab replicates are usually not done · Essentially, the analysis is a one-shot deal, you don’t get a second chance, so be careful · Field replicates should be done every 10 samples · Cut filters in half and save one half if nervous Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 79
Periphyton Photo for section change Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 80
Periphyton · Collection: · Qualitative grabs or scrapings versus quantitative sampling from a known surface area · Different methods are used for collecting periphyton from rocks, woody debris, macrophytes, bottom substrates or other substrates Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 81
Periphyton – in situ sampling · Resulting material from a rock scrub (to the right) containing: · Macro invertebrates · Detritus · Fungi · Bacteria · as well as algae Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 82
Periphyton – sample prep Here’s a portion of the previous sample after being deposited on a glass fiber filter in preparation for chlorophyll extraction or AFDW determination. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 83
Periphyton – biomass estimation · Wet weight · Dry weight (dried at 103– 105 o C) · Ash free dry weight (AFDW) · Loss on ignition (LOI) · Combust at 475 -550 o C Muffle furnace · Chlorophyll (extract as per phytoplankton) · Particulate organic carbon and/or nitrogen (POC or PON) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 84
Periphyton – biomass calculations · Once you have a measure of chlorophyll or AFDW you’ll need to calculate per unit area. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 85
Periphyton biovolume · Measure cell dimensions with an ocular or stage micrometer to calculate cell volume. B A B Rod A A Sphere Developed by: Axler, Ruzycki Ellipsoid Updated: Dec. 29, 2003 U 3 -m 9 a-s 86
Bacteria – E. coli and fecal coliforms Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 87
Bacteria – E. coli and fecal coliforms · Fecal bacteria are used as indicators of possible sewage contamination · These bacteria indicate the possible presence of disease-causing bacteria, viruses, and protozoans that also live in human and animal digestive systems · E. coli is currently replacing the fecal coliform assay in most beach monitoring programs Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 88
Bacteria - indicators · The most commonly-tested fecal bacteria indicators are: · total coliforms · fecal coliforms · Escherichia coli (E. coli) · fecal streptococci · and enterococci · All but E. coli include several species of bacteria · E. coli is a single species in the fecal coliform group Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 89
Bacteria – EPA standards The U. S. EPA recommended standard for E. coli concentration in recreational waters: · The geometric mean for > 5 samples within a 30 -day period shall not >126 E. coli colonies per 100 ml of water; and · No sample > 235 E. coli colonies/100 ml of water in a single sample For fecal coliforms: · Geometric mean for > 5 samples within a 30 -day period not > 200 cfu/100 m. L · < 10 % of samples > 400 cfu/100 m. L in any 30 -day period Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 90
Bacteria – 2 indicator methods Two basic methods: 1. membrane filtration 2. multiple-tube fermentation http: //picturethis. pnl. gov/picturet. nsf/f/uv? open&SMAA-3 V 9 T 37 Developed by: Axler, Ruzycki http: //www. intelligence. gov/2 community_examples. shtml Updated: Dec. 29, 2003 U 3 -m 9 a-s 91
Bacteria – membrane filter technique · The fecal coliform MF procedure uses an enriched lactose medium and incubation temperature of 44. 5 ± 0. 2 o C for selectivity. · Results in 93% accuracy (APHA 1995) in differentiating between coliforms found in the feces of warm-blooded animals and those from other environmental sources. · Fecal Coliform is reported as colony forming units per 100 m. L (CFU/100 m. L). Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 92
Bacteria – membrane filter equipment · Materials needed for MF method: · Air incubator or water bath · Non-corrugated forceps · Heat sterilizer (Bacti. Cinerator) · Filter flask and tower (Autoclavable) · Vacuum pump or water aspirator Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 93
Bacteria – membrane filter equipment · MF materials (continued): · Sterile 50 mm petri plates (with tight-fitting lids) · Sterile 0. 45 um gridded membrane filters · Sterile absorbent pads · Autoclave (121 o C at 15 -17 psi) Developed by: Axler, Ruzycki http: //www. nbtc. cornell. edu/biofacility/autoclave. html Updated: Dec. 29, 2003 U 3 -m 9 a-s 94
Bacteria – membrane filter procedure Procedure: · Saturate the absorbent pad with M-FC broth · Select a sample volume that will yield 20 -60 colonies/filter · Filter sample and dilution water through pad · Place pad into petri dish · Invert plates and place in incubator for 24 hrs Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 95
Bacteria – membrane filter counting · Fecal coliform colonies bacteria are various shades of blue. · Non-fecal colonies are gray to cream colored. · normally, few of these are present. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 96
Bacteria – MF counting (cont. ) http: //water. usgs. gov/owq/Field. Manual/Chapter 7. 1/images/Fig 7. 1 -3. gif image showing method of counting Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 97
Bacteria – multiple tube fermentation MTF image process http: //water. usgs. gov/owq/Field. Manual/Chapter 7. 1/images/Fig 7. 1 -3. gif Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 98
Bacteria – cleaning and sterilizing All equipment Wash equipment thoroughly with dilute nonphosphate, laboratory-grade detergent. Rinse 3 X with hot tap water Rinse again 3 -5 X with deionized or glass-distilled water. Glass, polypropylene, or Teflon™ bottles If sample will contain residual chlorine or other halogens, add Na 2 S 2 O 3. If sample will contain > 10 ug/L trace elements, add EDTA. Autoclave at 121 C for 15 min or bake glass jars at 170 C for 2 hrs. Stainless-steel field units Flame sterilize with methanol (Millipore™ Hydrosol units only), or autoclave, or bake at 170 C for 2 hrs Portable submersible pumps and pump tubing Autoclavable equipment (preferred): autoclave at 121 C for 15 min. Non-autoclavable equipment: Submerge sampling system in a 200 mg/L laundry bleah solution and circulate solution through pump and tubing for 30 min; follow with thorough rinsing, inside and out, with sample water pumped from the well. **SEE NOTES Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 99
Bacteria – USGS summary Test (media type) Ideal count range (colonies per filter) Total coliform bacteria (m-Endo) Typical colony color, size, and morphology 20 -80 Colonies are round, raised and smooth; 1 to 4 mm di; and red with golden-green metallic sheen. Escherichia coli After primary culture as total coliform colonies on m-Endo (NA-MUG) None given but much fewer in number than total coliforms on the same filter Colonies are cultured on m-Endo media as total coliform colonies. After incubation on NA-MUG, colonies have blue florescent margins with a dark center. Count under a long wave ultra violet lamp in a completely dark room. Fecal coliform bacteria (m-TEC) 20 -60 Colonies are round, raised and smooth with even to lobate margins; 1 to 6 mm di; light to dark blue in whole or part. Some may have brown or cream colored centers. Escherichia coli (m-TEC) 20 -80 Colonies are round, raised and smooth; 1 to 4 mm di; yellow to yellow brown; many have darker raised centers. Fecal streptococci (KF media) 20 -100 Colonies are small, raised, and spherical; about 0. 5 to 3 mm di; glossy pink or red in color. Enterococci (m-E and EIA) 20 -60 Colonies are round, smooth and raised; 1 to 6 mm di; pink to red with a black or red dish – brown precipitate on underside. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 100
Fecal coliforms – troubleshooting poor seal around the edges; poorly seated with air bubble Uneven; not mixed well; low volume Dry spot from poor seating No matter which assay is used, after incubation there should be ~20 -60 colonies evenly distributed across the Petri dish Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 101
Fecal coliforms – troubleshooting (cont. ) Too many – use less sample Too few – use more sample Looks good Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 102
Aquatic vegetation Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 103
Aquatic vegetation – biomass method · Harvested material is sorted by species · Stripped of periphyton · Weighed, dried at 103 -105 o C and reweighed · Biomass is usually expressed as wet weight or dry weight per m 2 · Dried material may be ground and subsampled for organic matter, %C, %N, %P or other constituents Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 104
Aquatic vegetation – biomass method · A separate set of carefully pressed and dried specimens may be set aside for archives · A blotted, but wet subsample may be extracted for chlorophyll. · The wet: dry ratio is important for comparing areal chlorophyll values to other parameters Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 105
Zooplankton Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 106
Zooplankton – sample preservation · Most commonly 95% ethanol or 5% formaldehyde (formalin) · Animals preserved in formalin sometimes become distorted which complicates size measurements. · One solution involves the addition of 40 g/L sucrose to the 5% formaldehyde. · Rose Benegal dye is also used by many to stain the critters for ease of identification Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 107
Zooplankton – equipment 1. 6. Hensen Stemple pipettes 2. 5. Folsom 3. Plankton Splitter Sedgwick-Rafter counting slide 4. Compound microscope All B/W images from Wild. Co. com Dissecting microscope Ward Counting Wheel Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 108
Zooplankton – taxonomy · Taxonomy is complex so ID to species level is best left to the experts but genus and order level are relatively easy · As with phytoplankton, organism size is important to determine http: //biology. usgs. gov/s+t/SNT/noframe/mr 181 f 06. htm Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 109
Zooplankton – detailed biomass Daphnia pulex Approximate sizes (not to scale) Cyclops 1 mm 2 mm 0. 5 mm Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 110
Zooplankton –total biomass · Total community biomass may be estimated by simply measuring the wet weight (or dry weight) of the zoops from a given tow with known volume. Leptadora http: //www. glaquarium. org/ Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 111
Zooplankton – biomass example · To determine # animals/L you need to determine the volume of water filtered through the net. Example Using a Wisconsin net with a small, 13 cm diameter opening for a 0 to 5 m vertical tow: Where d = 0. 13 m and z = 5. 0 m = 0. 66 m 3 = 66 liters Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 112
Benthic samples Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 113
Benthic samples · Processing benthic invertebrate samples · Determining sediment bulk characteristics: · Texture (% sand, silt, clay) · % organic matter · Total carbon, nitrogen, and phosphorus concentration · Sediment oxygen demand Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 114
Benthic invertebrates – sample processing · Sorting into taxonomic groups, · Identifying to desired taxonomic level, · Data entry http: //www. anr. state. vt. us/dec/waterq/bassmacro. htm Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 115
Benthic invertebrates – sample processing · Rinse the sample in a 500 m mesh sieve to remove and fine sediment. · Sticks and leaves can be visually inspected and then discarded. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 116
Benthic invertebrates - sub sampling · Spread the sample evenly across a pan marked with grids · Randomly select 4 squares, remove the material and preserve in jars Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 117
Benthic invertebrates – identification · Most organisms are identified to the lowest possible taxonomic level · Lowest taxonomic level depends on the goals of the analysis, expertise, and available funds Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 118
Benthic invertebrates – data processing · Metric · An attribute with empirical change in value along a gradient of human influence · In other words, a measurement made to determine if humans have had an impact in a natural system. · Index · An integrative expression of site conditions across multiple metrics. An index of biological integrity is often composed of at least 7 metrics. (Karr and Chu 1997) Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 119
Benthic invertebrates - data metrics · Many metrics have been developed for aquatic invertebrates. Richness measures Composition measures Tolerance measures Trophic/habitat measures Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 120
Benthic sediment – bulk properties Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 121
Sediment - bulk properties · Texture · % organic matter · Total carbon · Organic matter · Nutrient content: · Bioavailable phosphorus · Total nitrogen · Sediment oxygen demand Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 122
Sediment - texture · Refers to the shape, size, and three-dimensional arrangement of the particles that make up sediment · Gravels and pebbles can be measured using calipers · Sand is measured using sieves of different mesh size · Silts and clays are more difficult Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 123
Sediment - % organic matter · Measured as mg/g sediment · % carbon may also be important to measure, particularly in studies of sediments contaminated with pesticides, PAHs, and dioxide Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 124
Sediment – phosphorus content · Potentially bioavailable P from sediment or sediment traped material can be estimated from a single extraction with 0. 1 N Na. OH. · Total P can be extracted using persulfate or hot HCl acid procedure. · Both procedures involve extracting P into a solution which is then analyzed for P content using the ortho-P ascorbic acid method. Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 125
Sediment – C: N content · Coming soon Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 126
Sediment – exchangeable NH 4+ · Coming soon Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 127
Sediment – oxygen demand · Coming soon Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U 3 -m 9 a-s 128
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