Transcript Mod9 Intro to Lake Surveys: Lab Techniques

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
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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
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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
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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
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Alkalinity and hardness
Photo of pH test
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
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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
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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
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Alkalinity – analysis
 pH meter
 Buret*
 Thermometer
 Magnetic stirrer and
stir bar
 Top loading balance
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
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Alkalinity- analysis
(2 B  C )  N  50000
total alkalinity, m g CaCO3 / L 
m Lsam ple
or
(2 B  C )  N  999100
total alkalinity,  eq / L 
m Lsam ple
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
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Hardness – analysis
 Hardness is, ideally, determined by calculation from the
separate determinations of calcium and magnesium.
Hardness, in units of mg CaCO3/L
 2.497[Ca]  4.118[Mg]
Where Ca and Mg are in mg/L
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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Alkalinity and hardness – analysis
There are also titration test
kits available for both
alkalinity and hardness
www.lamotte.com
www.hach.com
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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Nutrients
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
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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
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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
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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
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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
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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
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Color measuring instruments
Hach DR2400 portable
spectrophotometer
Developed by: Axler, Ruzycki
•Bausch & Lomb
spectrophotometer 20
Updated: Dec. 29, 2003
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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:
NH4-N and NO3-N:
KH24PO4,
K2aHPO4,
Use dried NH
NO3 as
dual
NaH2PO4(50%
or Naof
standard
each form)
2HPO4
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
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Standard calibration curves
NH4-N standards
Developed by: Axler, Ruzycki
Good straight line fit:
ABS = a + b*[Conc]
Updated: Dec. 29, 2003
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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
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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 2nd line from 12002000 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
The line becomes nonlinear after ABS ~ 1.0 (~
1000 ugN/L)
Updated: Dec. 29, 2003
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Absorbance @ 880 nm
Some data from northern Minnesota lakes
0.600
ABS = (-0.0010) + (0.00254)* P
Calibration curve
0.500
R2 = 0.9997 n=12
0.400
0.300
0.200
= std
Sample #1 = 11.2 ugP/L
0.100
0.000
0
50
100
150
200
ortho-P (ug/L)
Conclusion:
The data are valid
Developed by: Axler, Ruzycki
250
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% 
Updated: Dec. 29, 2003
U3-m9a-s28
Total suspended solids and turbidity
Sediment plume off the south shore of Lake Superior
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
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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
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Total suspended solids - method
1. Filter a known amount of
water through a pre-washed,
pre-dried (at 103-105oC), 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
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Total suspended solids - method
What type of filter to
use?
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
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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
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Total suspended solids
 Necessary TSS equipment
Analytical balance
Drying oven
Filter and petri dish
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
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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
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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-s39
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
http://www.bradwoods.org/eagles/turbidity.htm
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s40
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
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Turbidity – formazin standards
 Example of a set of formazin standards
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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Turbidity  Here is a range of NTUs using clay
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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
U3-m9a-s44
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-s45
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-s46
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
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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
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Biochemical Oxygen Demand (BOD)
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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-s50
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
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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-s52
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
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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
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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
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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
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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-s57
BOD
Calculations
 When dilution water is not seeded:
D1  D 2
BOD 5day(mg / L) 
P
 When dilution water is seeded:
( D1  D 2)  ( B1  B 2) f
BOD 5day(mg / L) 
P
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s58
Phytoplankton/Algae – counting methods
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s59
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-s60
Algae – counting methods
Microscopes capable of magnifications of 100X to 1000X
Compound
microscope
Inverted microscope
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
Less expensive
inverted microscope
U3-m9a-s61
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
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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
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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-s64
Algae – determining biovolume
Some formulas to estimate biovolume from cell
dimensions (Wetzel & Likens 2000)
B
A
A
B
A
Rod
Sphere
Ellipsoid
A / 6
AB2 / 6
AB / 4
2
Developed by: Axler, Ruzycki
3
Updated: Dec. 29, 2003
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Algae – chlorophyll determination
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
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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-s67
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-s68
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-s69
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-s70
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-s71
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-s72
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-s73
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-s74
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-s75
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-s76
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
U3-m9a-s77
Algae –spectrophotometry calculations
26.7[ E  E ]  V
chlorophyll a( g / L) 
V L
26.7[1.7 E  E ]  V
phaeophytin( g / L) 
V L
664 b
665 a
ext
sample
665 b
664 a
ext
sample
Where:
b = before acidification
a = after acidification
E664b - [{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)
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s78
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-s79
Periphyton
Photo for section change
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s80
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-s81
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-s82
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-s83
Periphyton – biomass estimation
 Wet weight
 Dry weight (dried at 103–105o C)
 Ash free dry weight (AFDW)
 Loss on ignition (LOI)
 Combust at 475-550o 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
U3-m9a-s84
Periphyton – biomass calculations
 Once you have a measure of chlorophyll or AFDW
you’ll need to calculate per unit area.
Project Name
Water Quality Samples
2002
NRRI Central Analytical Lab
emr 12/4/02
Periphyton
Whatever Creek
Whatever Creek
scrub area = 2.3cmX3.5cm=8cm2
8 cm2=0.0008 m2 X 3 scrubs = .0024 m2 total area
Sample
Run
chlorophyll phaeophytin
Date
Date
ug/L
ug/L
5/6/2002 5/15/2002
130
60
Sample
Run
Date
Date
5/6/2002
5/8/2002
Developed by: Axler, Ruzycki
Dry Wt
mg/L
156
AFDW
mg/L
117
chlorophyll
volume
total
chlorophyll
filtered (mLs) volume (mLs) mg/m2
45
45
2.4
AFDW
total
volume
volume (mLs) filtered (mLs)
319
122
Updated: Dec. 29, 2003
AFDW
g/m2
6.0
U3-m9a-s85
Periphyton biovolume
 Measure cell dimensions with an
ocular or stage micrometer to
calculate cell volume.
B
A
B
Rod
AB2 / 4
A
A
Sphere
A3 / 6
Developed by: Axler, Ruzycki
Ellipsoid
AB2 / 6
Updated: Dec. 29, 2003
U3-m9a-s86
Bacteria – E. coli and fecal coliforms
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s87
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-s88
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-s89
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-s90
Bacteria – 2 indicator methods
Two basic methods:
1. membrane filtration 2. multiple-tube fermentation
http://picturethis.pnl.gov/picturet.
nsf/f/uv?open&SMAA-3V9T37
Developed by: Axler, Ruzycki
http://www.intelligence.gov/2community_examples.shtml
Updated: Dec. 29, 2003
U3-m9a-s91
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-s92
Bacteria – membrane filter equipment
 Materials needed for MF
method:
 Air incubator or water
bath
 Non-corrugated forceps
 Heat sterilizer (BactiCinerator)
 Filter flask and tower
(Autoclavable)
 Vacuum pump or water
aspirator
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s93
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 1517 psi)
Developed by: Axler, Ruzycki
http://www.nbtc.cornell.edu/biofacility/autoclave.html
Updated: Dec. 29, 2003
U3-m9a-s94
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-s95
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-s96
Bacteria – MF counting (cont.)
http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
image showing method of counting
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s97
Bacteria – multiple tube fermentation
MTF image process
http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s98
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 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-s99
Bacteria – USGS summary
Test (media type)
Ideal count range
(colonies per filter)
Total coliform bacteria
(m-Endo)
20-80
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
Typical colony color, size, and morphology
Colonies are round, raised and smooth; 1 to 4 mm di; and
red with golden-green metallic sheen.
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-s100
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
U3-m9a-s101
Fecal coliforms – troubleshooting (cont.)
Too many – use
less sample
Too few –
use more sample
Looks good
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s102
Aquatic vegetation
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s103
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-s104
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-s105
Zooplankton
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s106
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-s107
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 WildCo.com
Dissecting
microscope
Ward Counting
Wheel
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s108
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.ht
m
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s109
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
U3-m9a-s110
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
U3-m9a-s111
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:
volum e(m3 ) 
d 2
4
 volume (m ) 
3
z

Where d = 0.13 m
and z = 5.0 m

0.13  5.0
4
Developed by: Axler, Ruzycki
2
= 0.66 m3
= 66 liters
Updated: Dec. 29, 2003
U3-m9a-s112
Benthic samples
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s113
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-s114
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-s115
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-s116
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-s117
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-s118
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-s119
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-s120
Benthic sediment – bulk properties
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s121
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-s122
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-s123
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-s124
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-s125
Sediment – C:N content
 Coming soon
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s126
Sediment – exchangeable NH4+
 Coming soon
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s127
Sediment – oxygen demand
 Coming soon
Developed by: Axler, Ruzycki
Updated: Dec. 29, 2003
U3-m9a-s128