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Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9.

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1 Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

2 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s2 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.

3 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s3 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.

4 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s4 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

5 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s5 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

6 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s6 Alkalinity and hardness Photo of pH test

7 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s7 Alkalinity and hardness - what is it? Alkalinity: a measure of the ability of a water sample to neutralize strong acid Expressed as mg CaCO 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 CaCO 3 per liter

8 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s8 Alkalinity and hardness - how to sample Usually collected at the surface in lakes (0 to 1m depth) Keep the sample cool (4 o C refrigerated) and out of direct sunlight

9 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s9 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 (CaCO 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.

10 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s10 Alkalinity – analysis pH meter Buret* Thermometer Magnetic stirrer and stir bar Top loading balance

11 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s11 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 pH (known as an endpoint) At the endpoint pH, 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 CaCO3) The value may also be reported in milliequivalents by dividing by 50

12 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s12 Alkalinity- analysis or Where: B = mL titrant first recorded pH (i.e., to pH = 4.5) C = total mL titrant to reach pH 0.3 unit lower, and N = normality of acid (titrant)

13 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s13 Hardness – analysis Hardness is, ideally, determined by calculation from the separate determinations of calcium and magnesium. Where Ca and Mg are in mg/L Hardness, in units of mg CaCO 3 /L

14 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s14 Alkalinity and hardness – analysis There are also titration test kits available for both alkalinity and hardness

15 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s15 Nutrients

16 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s16 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.

17 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s17 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

18 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s18 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

19 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s19 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 2. Prepare calibration standards - solutions with a range of nutrient concentrations 3. Compare sample absorbances to calibration standard absorbances to estimate sample concentrations

20 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s20 Nutrients: colorimetry & spectrophotometry 4. Add reagents to develop color 5. Compare using a chart or color wheel using a colorimeter determining the absorbance using a spectrophotometer Low ….…. to ……. High Phosphate concentration

21 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s21 Color comparators and colorimetry Test Kits – There are many brands available Images from Color TubeColor DiscPocket Colorimeter

22 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s22 Color measuring instruments Hach DR2400 portable spectrophotometer Bausch & Lomb spectrophotometer 20

23 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s23 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: Use dried KH 2 PO4, K 2 HPO4, NaH 2 PO4 or Na 2 HPO4 NH 4 -N and NO 3 -N: Use dried NH 4 NO 3 as a dual standard (50% of each form)

24 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s24 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

25 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s25 Standard calibration curves NH 4 -N standards Good straight line fit: ABS = a + b * [Conc]

26 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s26 N Estimating concentrations So, if sample #3 had an absorbance of 0.290… Its concentration would be ~ 0.33 ppm N …

27 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s27 #2 Standard curves – troubleshooting The line becomes non- linear after ABS ~ 1.0 (~ 1000 ugN/L) Errors in preparing the 0.25 and 0.50 ppm standards perhaps ? Example #1 – Live with it or re-run the batch #1 Example #2 – Fit a straight line from and a 2 nd line from ugN/L Use non-linear quadratic instead of a line for ugN/L Re-read in smaller cuvette or dilute and re-run

28 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s28 Some data from northern Minnesota lakes Calibration curve = std ABS = ( ) + ( )* P R2 = n=12 Sample #1 = 11.2 ugP/L Sample #1 - Replicate = 12.6 ugP/L Sample # Spike = 59.4 ugP/L % RPD = 100* (1.4)/ 11.9 = 12% % R = 100* ( )/50 = 95% Conclusion: The data are valid

29 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s29 Total suspended solids and turbidity Sediment plume off the south shore of Lake Superior

30 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s30 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

31 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s31 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)

32 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s32 Total suspended solids - method 1.Filter a known amount of water through a pre-washed, pre-dried (at o C), pre- weighed (~ 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

33 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s33 Total suspended solids - method What type of filter to use?

34 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s34 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

35 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s35 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

36 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s36 Necessary TSS equipment Drying oven Analytical balance Filter and petri dish Total suspended solids

37 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s37 Calculate TSS by using the equation below: Total suspended solids 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)

38 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s38 How do turbidity and TSS relate? A general rule of thumb: 1 mg TSS/L ~ NTUs of turbidity BUT – Turbidity scattering depends on particle size so this is only a rough approximation

39 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s39 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)

40 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s40 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

41 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s41 Turbidity – units and reporting Nephelometric Turbidity Units (NTU) Standards are formazin or other certified material JTUs are from an older technology in which a candle flame was viewed through a tube of water 1 NTU = 1 JTU (Jackson Turbidity Unit)

42 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s42 Turbidity – formazin standards Example of a set of formazin standards

43 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s43 Turbidity - Here is a range of NTUs using clay

44 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s44 Bench and portable instruments and kits vs. YSI wiping turbidity YSI 6820 with unwiped turbidity Hydrolab Turbidity – meters and probes Submersible Turbidimeters

45 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s45 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.

46 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s46 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

47 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s47 Turbidity - standards Standards range depends on anticipated sample values Lakes - typically 0-20 NTU Streams and wetlands , 0-50 or 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

48 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s48 Table of standards Prepare daily2 to 20 NTUHach Company Prepare weeklyAll dilutionsEPA Region 5 Prepare dailyAll dilutionsStandard Methods (APHA 1995) Prepare monthly20 to 40 NTU Suggested holding timesConcentrationsSource Turbidity – standards

49 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s49 Biochemical Oxygen Demand (BOD)

50 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s50 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)

51 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s51 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 pH the presence of certain kinds of microorganisms the type of organic and inorganic material in the water

52 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s52 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

53 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s53 BOD - analysis Equipment needed: Incubation bottles Air incubator or water bath thermostatically controlled at 20 +/- 1 o C DO meter and probe

54 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s54 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

55 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s55 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

56 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s56 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

57 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s57 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

58 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s58 BOD Calculations When dilution water is not seeded: When dilution water is seeded:

59 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s59 Phytoplankton/Algae – counting methods

60 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s60 Algae- counting methods Wet mounts Filter Counting chambers Utermohl requires an inverted microscope (light from above) Sedgewick rafter chamber Hemocytometer

61 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s61 Microscopes capable of magnifications of 100X to 1000X Inverted microscope Compound microscope Less expensive inverted microscope Algae – counting methods

62 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s62 Algae- taxonomy Use an algal taxonomic key that shows species from your geographical area Phytoplankton are continually being described and re-classified so its essential for a good taxonomist to keep current (not easy by any means) Its a good idea to take photographs of slides for cataloging

63 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s63 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

64 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s64 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

65 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s65 Algae – determining biovolume Some formulas to estimate biovolume from cell dimensions (Wetzel & Likens 2000) Rod B A Sphere A Ellipsoid B A

66 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s66 Algae – chlorophyll determination

67 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s67 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

68 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s68 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

69 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s69 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

70 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s70 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

71 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s71 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

72 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s72 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 mL Corex centrifuge tubes with caps

73 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s73 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 MgCO 3 solution near the end When all the water has been pulled through, fold the filter into quarters and wrap in foil

74 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s74 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

75 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s75 Algae – chlorophyll extraction & analysis Chlorophyll extraction: Tear filter into several pieces Place in a test tube Add 10 mLs of 90% acetone Extract overnight at 4 o C Chlorophyll analysis: After hr extraction, centrifuge to settle filter debris Read absorbance or fluorescence of the supernatant

76 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s76 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)

77 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s77 Algae – chlorophyll and phaeophytin What is phaeophytin? Degradation product of chlorophyll Absorbance wavelength (665 nm) is very close to that of chlorophyll (664 nm) acid H

78 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s78 Where: b = before acidification a = after acidification E 664b - [{Abs 664b(sample) –Abs 664b(blank) }-{Abs 750b(sample) –Abs 750b(blank) }] E 665a - [{A 665a(sample) -Abs665a(blank)}-{Abs 750a(sample) -Abs 750a(blank) }] V ext = Volume of 90% Acetone used in the extraction (mL) V sample = Volume of water filtered (L) L = Cuvette path length (cm) Algae –spectrophotometry calculations

79 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s79 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 dont 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

80 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s80 Photo for section change Periphyton

81 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s81 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

82 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s82 Periphyton – in situ sampling Resulting material from a rock scrub (to the right) containing: Macro invertebrates Detritus Fungi Bacteria as well as algae

83 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s83 Periphyton – sample prep Heres a portion of the previous sample after being deposited on a glass fiber filter in preparation for chlorophyll extraction or AFDW determination.

84 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s84 Wet weight Dry weight (dried at 103–105 o C) Ash free dry weight (AFDW) Loss on ignition (LOI) Combust at o C Chlorophyll (extract as per phytoplankton) Particulate organic carbon and/or nitrogen (POC or PON) Periphyton – biomass estimation Muffle furnace

85 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s85 Once you have a measure of chlorophyll or AFDW youll need to calculate per unit area. Periphyton – biomass calculations

86 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s86 Periphyton biovolume Measure cell dimensions with an ocular or stage micrometer to calculate cell volume. Sphere A Ellipsoid B A Rod B A

87 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s87 Bacteria – E. coli and fecal coliforms

88 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s88 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

89 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s89 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

90 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s90 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/100mL 400 cfu/100 mL in any 30-day period

91 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s91 Bacteria – 2 indicator methods Two basic methods: 1. membrane filtration 2. multiple-tube fermentation community_examples.shtml nsf/f/uv?open&SMAA-3V9T37

92 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s92 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 mL (CFU/100 mL).

93 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s93 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

94 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s94 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 psi)

95 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s95 Bacteria – membrane filter procedure Procedure: Saturate the absorbent pad with M-FC broth Select a sample volume that will yield colonies/filter Filter sample and dilution water through pad Place pad into petri dish Invert plates and place in incubator for 24 hrs

96 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s96 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.

97 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s97 image showing method of counting Bacteria – MF counting (cont.)

98 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s98 MTF image process Bacteria – multiple tube fermentation

99 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s99 Bacteria – cleaning and sterilizing All equipmentWash 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

100 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s100 Bacteria – USGS summary Test (media type)Ideal count range (colonies per filter) Typical colony color, size, and morphology Total coliform bacteria (m-Endo) 20-80Colonies 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) 20-60Colonies 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) 20-80Colonies are round, raised and smooth; 1 to 4 mm di; yellow to yellow brown; many have darker raised centers. Fecal streptococci (KF media) Colonies are small, raised, and spherical; about 0.5 to 3 mm di; glossy pink or red in color. Enterococci (m-E and EIA) 20-60Colonies are round, smooth and raised; 1 to 6 mm di; pink to red with a black or red dish – brown precipitate on underside.

101 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s101 No matter which assay is used, after incubation there should be ~20-60 colonies evenly distributed across the Petri dish poor seal around the edges; poorly seated with air bubble Dry spot from poor seating Uneven; not mixed well; low volume Fecal coliforms – troubleshooting

102 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s102 Too many – use less sample Too few – use more sample Looks good Fecal coliforms – troubleshooting (cont.)

103 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s103 Aquatic vegetation

104 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s104 Aquatic vegetation – biomass method Harvested material is sorted by species Stripped of periphyton Weighed, dried at 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

105 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s105 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

106 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s106 Zooplankton

107 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s107 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

108 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s108 Folsom Plankton Splitter 1. Hensen Stemple pipettes All B/W images from WildCo.com Sedgwick-Rafter counting slide Ward Counting Wheel 4. Compound microscope 6. Zooplankton – equipment Dissecting microscope

109 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s109 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 m

110 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s110 Cyclops 1 mm 2 mm 0.5 mm Approximate sizes (not to scale) Zooplankton – detailed biomass Daphnia pulex

111 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s111 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

112 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s112 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: Zooplankton – biomass example Where d = 0.13 m and z = 5.0 m = 0.66 m 3 = 66 liters

113 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s113 Benthic samples

114 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s114 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

115 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s115 Benthic invertebrates – sample processing Sorting into taxonomic groups, Identifying to desired taxonomic level, Data entry

116 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s116 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.

117 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s117 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

118 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s118 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

119 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s119 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)

120 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s120 Benthic invertebrates - data metrics Many metrics have been developed for aquatic invertebrates. Richness measures Composition measures Tolerance measures Trophic/habitat measures

121 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s121 Benthic sediment – bulk properties

122 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s122 Sediment - bulk properties Texture % organic matter Total carbon Organic matter Nutrient content: Bioavailable phosphorus Total phosphorus Total nitrogen Sediment oxygen demand

123 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s123 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

124 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s124 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 Measured as mg/g sediment % carbon may also be important to measure, particularly in studies of sediments contaminated with pesticides, PAHs, and dioxide

125 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s125 Sediment – phosphorus content Potentially bioavailable P from sediment or sediment traped material can be estimated from a single extraction with 0.1 N NaOH. 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.

126 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s126 Sediment – C:N content Coming soon

127 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s127 Sediment – exchangeable NH 4 + Coming soon

128 Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s128 Sediment – oxygen demand Coming soon

129


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