introduction to lake surveys: laboratory techniques unit 3: module 9
TRANSCRIPT
Introduction to Lake Surveys: Laboratory Techniques
Unit 3: Module 9
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.
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.
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
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s6
Alkalinity and hardness
Photo of pH test
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 CaCO3 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 CaCO3 per liter
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 (4oC refrigerated) and out of direct sunlight
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
(CaCO3) 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 U3-m9a-s10
Alkalinity – analysis
pH meter Buret* Thermometer Magnetic stirrer and
stir bar Top loading balance
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s11
Alkalinity- analysis
Reagents 0.04 N H2SO4 (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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s12
Alkalinity- analysis
samplemL
NCBLCaCOmgalkalinitytotal
50000)2(/, 3
samplemL
NCBLeqalkalinitytotal
999100)2(/,
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)
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 CaCO3/L
][118.4][497.2 MgCa
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
www.hach.com
www.lamotte.com
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s15
Nutrients
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.
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
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
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
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s21
Color comparators and colorimetry
Test Kits – There are many brands available
Images from www.hach.com
Color Tube Color Disc Pocket Colorimeter
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s22
Color measuring instruments
Hach DR2400 portable spectrophotometer
•Bausch & Lomb spectrophotometer 20
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 KH2PO4, K2HPO4,
NaH2PO4 or Na2HPO4
NH4-N and NO3-N:
Use dried NH4NO3 as a dual
standard (50% of each form)
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s25
Standard calibration curves
NH4-N standards
Good straight line fit:
ABS = a + b*[Conc]
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 …
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 0-1000 and a 2nd line from 1200-2000 ugN/L
Use non-linear quadratic instead of a line for 0-2000 ugN/L
Re-read in smaller cuvette or dilute and re-run
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s28
Some data from northern Minnesota lakes
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0 50 100 150 200 250
ortho-P (ug/L)
Ab
sorb
ance
@ 8
80 n
m Calibration curve
= std
ABS = (-0.0010) + (0.00254)* P
R2 = 0.9997 n=12
Sample #1 = 11.2 ugP/L
Sample #1 - Replicate = 12.6 ugP/L
Sample #1 + 50 Spike = 59.4 ugP/L
% RPD = 100* (1.4)/ 11.9 = 12%
% R = 100* (59.4-11.9)/50 = 95%
Conclusion:
The data are valid
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
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
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 4oC (but the sooner the better)
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 103-105oC), pre-
weighed (~ + 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 U3-m9a-s33
Total suspended solids - method
What type of filter to use?
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
www.whatman.com
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
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
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)
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 ~ 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 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)
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s40
http://www.bradwoods.org/eagles/turbidity.htm
Turbidity
Turbidimeters Nephelometric optics
• nephelometric turbidity is estimated by using the scattering effect suspended particles have on light
• detector is at 90o from the light source
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
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 U3-m9a-s42
Turbidity – formazin standards
Example of a set of formazin standards
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s43
Turbidity -
Here is a range of NTUs using clay
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
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.
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
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 - 0-20, 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 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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s49
Biochemical Oxygen Demand (BOD)
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 20o C)
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 20o 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
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 4o C Warm chilled samples to 20o C before analysis
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 +/- 1o C
DO meter and probe
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
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s56
BOD - procedure
Blanks Prepare dilution water, bring to 20o 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 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 20o C Read final DO Calculate BOD5 correcting for the exact duration
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s58
BOD
Calculations When dilution water is not seeded:
When dilution water is seeded:
P
DDLmgBOD day
215 )/(
P
fBBDDLmgBOD day
)()()/(
21215
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s59
Phytoplankton/Algae – counting methods
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s61
Microscopes capable of magnifications of 100X to 1000X
Inverted microscopeCompound microscope
Less expensive inverted microscope
Algae – counting methods
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 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 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
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
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
4/2AB
B
A
6/3ASphere
A
Ellipsoid
6/2AB
B
A
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s66
Algae – chlorophyll determination
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
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
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
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
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
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
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 MgCO3 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 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 < 20o C
EPA holding time for a frozen chlorophyll filter is 2 weeks
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 4oC
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 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)
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s78
Where:
b = before acidificationa = after acidificationE664b - [{Abs664b(sample)–Abs664b(blank)}-{Abs750b(sample)–Abs750b(blank)}] E665a - [{A665a(sample)-Abs665a(blank)}-{Abs750a(sample)-Abs750a(blank)}] Vext = Volume of 90% Acetone used in the extraction (mL) Vsample = Volume of water filtered (L) L = Cuvette path length (cm)
Algae –spectrophotometry calculations
LV
VEELgalchlorophyl
sample
extab
][7.26
)/(665664
LV
VEELgnphaeophyti
sample
extab
]7.1[7.26
)/(664665
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
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 U3-m9a-s80
Photo for section change
Periphyton
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
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s83
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 U3-m9a-s84
Wet weight
Dry weight (dried at 103–105o C)
Ash free dry weight (AFDW) Loss on ignition (LOI) Combust at 475-550o C
Chlorophyll (extract as per phytoplankton) Particulate organic carbon and/or nitrogen
(POC or PON)
Periphyton – biomass estimation
Muffle furnace
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s85
Once you have a measure of chlorophyll or AFDW you’ll need to calculate per unit area.
Project NameWater Quality Samples scrub area = 2.3cmX3.5cm=8cm2
2002 8 cm2=0.0008 m2 X 3 scrubs = .0024 m2 total areaNRRI Central Analytical Labemr 12/4/02
Sample Run chlorophyll phaeophytin volume total chlorophyllPeriphyton Date Date ug/L ug/L filtered (mLs) volume (mLs) mg/m2Whatever Creek 5/6/2002 5/15/2002 130 60 45 45 2.4
Sample Run Dry Wt AFDW total volume AFDWDate Date mg/L mg/L volume (mLs) filtered (mLs) g/m2
Whatever Creek 5/6/2002 5/8/2002 156 117 319 122 6.0
chlorophyll
AFDW
Periphyton – biomass calculations Periphyton – biomass calculations
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.
6/3A
Sphere
A
6/2AB
Ellipsoid
B
A
4/2AB
Rod
BA
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s87
Bacteria – E. coli and fecal coliforms
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
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
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 < 10 % of samples > 400 cfu/100 mL in any 30-day
period
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
http://www.intelligence.gov/2-community_examples.shtml
http://picturethis.pnl.gov/picturet.nsf/f/uv?open&SMAA-
3V9T37
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 ± 0.2o 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).
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
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 (121o C at 15-
17 psi)
http://www.nbtc.cornell.edu/biofacility/autoclave.html
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 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 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.
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s97
image showing method of countinghttp://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
Bacteria – MF counting (cont.)
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s98
MTF image process
http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
Bacteria – multiple tube fermentation
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s99
Bacteria – cleaning and sterilizing
All equipment Wash equipment thoroughly with dilute nonphosphate, laboratory-grade detergent.Rinse 3 X with hot tap waterRinse 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 Na2S2O3. 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 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)
20-80 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 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
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.)
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s103
Aquatic vegetation
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 103-105o C and reweighed Biomass is usually expressed as wet weight or
dry weight per m2
Dried material may be ground and subsampled for organic matter, %C, %N, %P or other constituents
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s106
Zooplankton
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
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
2.
5.
3.
Ward Counting Wheel
4.
Compound microscope
6.
Zooplankton – equipment
Dissecting microscope
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
http://biology.usgs.gov/s+t/SNT/noframe/mr181f06.htm
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
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.
http://www.glaquarium.org/
Leptadora
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
zd
mvolume 4
)(2
3 Where d = 0.13 mand z = 5.0 m
0.513.04
)( 23
mvolume = 0.66 m3
= 66 liters
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s113
Benthic samples
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
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
http://www.anr.state.vt.us/dec/waterq/bassmacro.htm
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.
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
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
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)
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
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s121
Benthic sediment – bulk properties
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
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
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
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.
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s126
Sediment – C:N content
Coming soon
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s127
Sediment – exchangeable NH4+
Coming soon
Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s128
Sediment – oxygen demand
Coming soon