introduction to lake surveys: laboratory techniques unit 3: module 9

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

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

Introduction to Lake Surveys: Laboratory Techniques

Unit 3: Module 9

Page 2: 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.

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

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.

Page 4: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 5: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 6: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s6

Alkalinity and hardness

Photo of pH test

Page 7: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 8: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

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

(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.

Page 10: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 11: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 12: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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)

Page 13: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 14: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 15: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Nutrients

Page 16: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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.

Page 17: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 18: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 19: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 20: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 21: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 22: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s22

Color measuring instruments

Hach DR2400 portable spectrophotometer

•Bausch & Lomb spectrophotometer 20

Page 23: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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)

Page 24: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 25: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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]

Page 26: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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 …

Page 27: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 28: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 29: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 30: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 31: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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)

Page 32: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 33: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s33

Total suspended solids - method

What type of filter to use?

Page 34: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 35: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 36: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 37: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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)

Page 38: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 39: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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)

Page 40: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 41: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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)

Page 42: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s42

Turbidity – formazin standards

Example of a set of formazin standards

Page 43: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s43

Turbidity -

Here is a range of NTUs using clay

Page 44: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 45: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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.

Page 46: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 47: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 48: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 49: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s49

Biochemical Oxygen Demand (BOD)

Page 50: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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)

Page 51: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 52: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 53: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 54: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 55: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 56: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 57: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 58: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 59: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s59

Phytoplankton/Algae – counting methods

Page 60: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 61: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 62: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 63: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 64: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 65: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 66: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

Developed by: Axler, Ruzycki Updated: Dec. 29, 2003 U3-m9a-s66

Algae – chlorophyll determination

Page 67: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 68: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 69: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 70: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 71: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 72: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 73: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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

Page 74: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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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

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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

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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)

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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

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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

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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

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Photo for section change

Periphyton

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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

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Periphyton – in situ sampling

Resulting material from a rock scrub (to the right) containing: Macro invertebrates Detritus Fungi Bacteria as well as algae

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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.

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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

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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

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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

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Bacteria – E. coli and fecal coliforms

Page 88: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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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

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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

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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

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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

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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).

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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

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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

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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

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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.

Page 97: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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image showing method of countinghttp://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif

Bacteria – MF counting (cont.)

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MTF image process

http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif

Bacteria – multiple tube fermentation

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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

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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.

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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

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Too many – use less sample

Too few – use more sample

Looks good

Fecal coliforms – troubleshooting (cont.)

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Aquatic vegetation

Page 104: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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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

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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

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Zooplankton

Page 107: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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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

Page 108: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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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

Page 109: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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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

Page 110: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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Cyclops

1 mm

2 mm

0.5 mm

Approximate sizes (not to scale)

Zooplankton – detailed biomass

Daphnia pulex

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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

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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

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Benthic samples

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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

Page 115: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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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

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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.

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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

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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

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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)

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Benthic invertebrates - data metrics

Many metrics have been developed for aquatic invertebrates.

Richness measures

Composition measures Tolerance measures

Trophic/habitat measures

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Benthic sediment – bulk properties

Page 122: Introduction to Lake Surveys: Laboratory Techniques Unit 3: Module 9

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Sediment - bulk properties

Texture % organic matter

Total carbon Organic matter

Nutrient content: Bioavailable phosphorus Total phosphorus Total nitrogen

Sediment oxygen demand

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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

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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

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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.

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Sediment – C:N content

Coming soon

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Sediment – exchangeable NH4+

Coming soon

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Sediment – oxygen demand

Coming soon

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