Biological Monitoring Manual

Authors Alliance for Aquatic Resource Monitoring

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The Alliance for Aquatic Resource Monitoring

Biological Monitoring
Manual

April 2009

© 2009 Alliance for Aquatic Resource Monitoring. Biological Monitoring
Manual. Is licensed under the Creative Commons Attribution-NonCommercial-
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Table of Contents
Background on ALLARM ................................................................................................................................ 3
Background on Manual ................................................................................................................................. 3
What is a Macroinvertebrate? ...................................................................................................................... 4
Method of Collection .................................................................................................................................... 4
Site Selection............................................................................................................................................. 4
Rocky Bottom Sampling ............................................................................................................................ 5
Muddy Bottom Sampling .......................................................................................................................... 7
Identification Categories ............................................................................................................................... 9
Sensitive .................................................................................................................................................... 9
Somewhat Sensitive ................................................................................................................................ 11
Tolerant ................................................................................................................................................... 13
Quantifying Your Results............................................................................................................................. 14
EASI Tally Sheet (Example) ...................................................................................................................... 15
Macroinvertebrate Survey (Example) ..................................................................................................... 16
EASI Tally Sheet ....................................................................................................................................... 17
Macroinvertebrate Survey ...................................................................................................................... 18
Glossary ....................................................................................................................................................... 20
References .................................................................................................................................................. 21
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Background on ALLARM:
The Alliance for Aquatic Resource Monitoring (ALLARM) is a project of the Environmental Studies
Department at Dickinson College. Since its founding in 1986, ALLARM has become a nationally
recognized technical and programmatic support center for community organizations interested in
watershed assessment, protection, and restoration. ALLARM program goals are to:

1) Enhance local action for the protection and restoration of
Pennsylvania watersheds by empowering communities with
scientific knowledge and tools to implement watershed
assessments;
2) Provide Dickinson College students with opportunities to
participate in community-based participatory research thereby
enhancing the quality of undergraduate science education; and
3) Be the leader in volunteer monitoring in Pennsylvania and a
national model for college-community partnerships.

Through the work of student and professional staff, ALLARM offers
comprehensive services to enable groups to use critical scientific tools to enhance environmental quality
and fully participate in community decision-making. The program staff includes a Director, an Assistant
Director, a faculty Science Director, and 12-14 undergraduate student staff.

For more information on please visit: www.dickinson.edu/allarm or email: allarm@dickinson.edu

Background on Manual:
The Environmental Alliance for Senior Involvement (EASI) developed a method for macroinvertebrate
assessment that enables watershed groups to easily monitor the long-term biological condition of
streams (Senior Environmental Corp). This is one of the few assessments that provide information
regarding impacts that continuously influence aquatic life. In addition to biological monitoring, it is
equally important to conduct chemical and visual assessments. It is critical to keep in mind that water
chemistry may not be the only influential factor on biological life. Since macroinvertebrates spend
almost their entire life in the stream, the condition of in-stream habitat is just as important as the
chemical condition of the water.

ALLARM uses this protocol to train volunteers on biological monitoring. This manual is a culmination of
tools and resources ALLARM has developed to complement training workshops using the EASI protocol.

Project funding provided by the Foundation for Pennsylvania Watersheds.
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What is a Macroinvertebrate?
Most macroinvertebrates are crustaceans or insect larvae that have no vertebral column (invertebrate)
and can be seen with the naked eye (macro). They spend the majority of their life living in streams or
other aquatic environments.

Macroinvertebrates are most abundant in riffle zones of streams where oxygen is more plentiful or in
areas around banks that provide more protection. Figure 1 shows examples of riffle zones and areas
with protective bank coverage that are likely to support healthy populations of macroinvertebrates. The
availability of food and oxygen along with chemical input will influence the type and abundance of
macroinvertebrates in these areas.

Figure 1. Section of stream illustrating natural
habitats for macroinvertebrates

Method of Collection:
In general, macroinvertebrate assessments should be conducted twice a year, once in the spring
(March-May) and once in the fall (August-October). Because of hatching and mating patterns, it is best
for northern areas of the state to conduct assessments in the earlier parts of the seasons while southern
areas should conduct them in the later parts of the seasons.

Site Selection:
In order to have a good idea of the macroinvertebrate population that would normally reside in a
stream, it is important to decide on a reference site that is the least impacted by pollution and has a
variety of habitats that will provide the greatest abundance and diversity of macroinvertebrates in that
area. For example, when investigating the impact of a discharge site, a site located upstream of the
discharge is typically used as a reference site since it is essentially “unimpacted” by the discharge. One
macroinvertebrate assessment consists of three sites within a 30-meter stretch of stream. Site #1 is the
most downstream location, #2 is the midstream reach, and #3 is the site furthest upstream. For each
location, you will thus have a total of three sites which will all be composited into one large sample. The
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three sites should contain riffle zones; however, different methods will need to be used depending on
the type of stream being sampled, for example rocky bottom versus muddy bottom.

Rocky Bottom Sampling:
This method is used for streams that have riffles and substrates
composed of gravel and/or cobble.

Necessary equipment:
o Measuring tape
o 1m X 1m, 500µm kick net or D-frame net
o Collection containers
o Forceps
o Magnifying glass/ hand lens
o Ethanol (to preserve macroinvertebrates if not identifying
same day as collection)
o Waders
o Fishing license if collecting >50 organisms

Procedure:
1. Identify the sampling area and mark off the 30-meter stretch you will be analyzing. Choose
three spots within that stretch. These three sites should be riffles areas and, if possible, should
have different flow velocities and substrate types in order to get a better representation of the
in-stream habitat diversity. It is often helpful to draw a sketch of the stream and label the three
sampling spots (#1 is downstream, #2 is midstream, and #3 is upstream). If you are using a
kicknet, proceed to step 2; if you are using a D-frame net, jump to step 6.

2. Always start at the downstream site first and also approach the location from downstream. This
prevents you from disturbing the other sites and the site you are about to sample with sediment
or other dislodged macroinvertebrates.

If you are using a kicknet, select your first 1 m X 1 m riffle area and have a team member
position the net, facing upstream. The net should be held at a 45 degree angle to the water’s
surface. However, depending on the stream depth, this angle can
be adjusted accordingly as long as water DOES NOT flow over the
net. This may result in the loss of macroinvertebrates! Also
ensure that the bottom of the net is fit to the bottom of the
stream. This can be done by using rocks from the sampling area
and anchoring the bottom of the net. Make sure to rub the rocks
either before or after kicking to remove any macroinvertebrates
that may be clinging to the rocks.

3. The second team member should stand in front of the kick net,
facing downstream. First pick up any large rocks in the 1m X 1m
area and rub them thoroughly in the water so that any
macroinvertebrates clinging to the rock will flow into the net. Be
sure to check for case-building caddisflies because they tend to
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stick securely to the rocks. Place the “clean” rocks outside of kicking area. Starting at the
upstream end of the 1m X 1m area, beginning kicking up the streambed to at least two or three
inches in depth. Once you reach the net, remove the rocks
that are anchoring the bottom of the net.

4. When removing the net, it is important to make sure water
does not flow over the top because it will take away some
of the organisms that you have just collected! The net
holder should grasp the top of the handles while the kicker
grabs the bottom handles and lift with a forward scooping
motion. You can then either pick directly from the net or
flush the contents into a bucket. Do not keep any fish,
amphibians, or reptiles. If you collect crayfish, it may be a
good idea to make note of them (because they are included
in the assessment) and release them as well.

If you intend to identify the macroinvertebrates later, put them in containers with enough
ethanol to cover all of the organisms. Make sure to label the containers according to which
location they are from.

5. Repeat steps 2 through 4 for the other two sites and combine all of the organisms from all three
sites. This will provide a better representation of the stream’s overall macroinvertebrate
community. Now continue on to step 10.

6. To sample with a D-frame net, you will complete 20 jabs in the 30 m stretch you have previously
selected. When using the jab method, samples should typically be taken from riffle areas, but
you should also get samples of the overall habitat in the stream. The majority of the jabs should
be taken from riffle areas (at least 10) and the remaining can be distributed among other stream
habitats.

7. Be sure to check the undersides of rocks in the area you a jabbing for any macroinvertebrates
that may be clinging to the rock, especially case-building caddisflies.

8. The contents from each jab can be dumped into
a large bucket with stream water, which will be
sorted later.

9. After completing 20 jabs, sort through all of the
debris for anything that moves and transfer the
organisms into the sorting trays.

10. When sorting, look for any movement and be
sure to check on all sides of leaves or other
debris for macroinvertebrates. The longer the
net is out of the water, the less movement you
will see so a magnifying glass may also be helpful
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to make sure all of the organisms are collected. Ice cube trays work well to sort the
macroinvertebrates into like-organisms.

11. Once you feel they are sufficiently sorted, use a hand lens and the following identification sheets
to identify the organisms to the Order level. If you plan to identify the organisms at a later
point, put them in a container with ethanol to help preserve them. Tally the number of
individuals in each Order on the field data sheet (attached). You are now ready to begin
calculating the stream water quality.

Muddy Bottom Sampling:
This method is used for streams that lack riffles and have muddy, silty, or sandy
bottoms.

Necessary Equipment:
o D-frame net
o Collection containers
o Forceps
o Ethanol (to preserve macroinvertebrates if not identifying same day as
collection)
o Fishing license if collecting >50 organisms

Procedure:
1. There are typically four types of habitat found in muddy bottom
streams: vegetated bank margins, snags and logs, aquatic vegetation
beds, and decaying organic matter. For muddy bottom streams, 20
“jabs” with a D-frame net in different habitat areas are combined to
get a representative sample of the macroinvertebrate population.
2. Note on your data sheet the types of habitats present and the number
of jabs taken from each. Some suggestions for distributing the jabs are
as follows:
a. If all for types are present, jab vegetated banks 10 times and divide the other 10 among
remaining habitats.
b. If three types are present, jab the least productive habitat (silt/sand/gravel) 5 times and
divide the remaining 15 between the more productive habitats.
c. If only two types are present, the silt/sand/gravel will likely be the most abundant, so
jab it 5 times and the more productive habitat 15 times.
d. If some habitats are plentiful and others are sparse, sample what you can of the sparse
habitats and take the remaining jabs of the most productive habitat available.
3. Begin sampling from the furthest downstream site and work upstream. The four habitat types
have different ways to collect the macroinvertebrates:
a. Vegetated Bank Margins—keeping the net underwater, jab vigorously with an upward
motion, brushing against vegetation and roots.
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b. Snags and Logs—holding the net under the submerged wood, use your other hand
(which should be gloved) to rub about 1 ft2 of the log. This equals one jab.
c. Aquatic Vegetation Beds—jab vigorously with an upward motion through the plant bed
d. Silt/sand/gravel—push the net forward about 1 foot upstream to dislodge the first few
inches of substrate. To avoid a netful of mud, you can sweep the net through the water
(as long as water does not run over the top of the net) to filter out some of the
sediments.
4. The contents from each jab can be dumped into a large bucket with stream water, which will be
sorted later.
5. After completing 20 jabs, sort through all of the debris for anything that moves and transfer the
organisms into the sorting trays.
6. Once you feel that you have found everything, use a hand lens and the following identification
sheets to identify the organisms to the Order level. If you plan to identify the organisms at a
later point, put them in a container with ethanol to help preserve them. Tally the number of
individuals in each Order on the field data sheet (attached). You are now ready to begin
calculating the stream water quality.
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Identification Categories:
Sensitive:

Water Penny Larvae—Order Coleoptera Mayfly Nymph—Order Ephemeroptera

 Wing pads absent  6 legs
 Flattened disc-like body  3 tails
 6 legs under dorsal plate  Gills on abdomen
 Single claw on end of legs

Meyers, 2009
Clapp, 2006

Gilled Snail—Class Gastropoda
Dobsonfly Larvae (Hellgrammite)—Order
Megaloptera  Vary in size
 Presence of operculum is
 6 legs characteristic of gilled snail
 8 abdominal segments each with a (seals the opening to the shell
filament when the foot is retracted)
 2 anal prolegs with hooks  Opening is on right hand side
 Well developed chewing parts
 Poorly developed eyes

Neuswanger, 2009 Lake County, Ohio, 2009
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Riffle Beetle—Order Coleoptera Non-Netspinning Caddisfly Larvae—Order
Trichoptera
 Oval elongate body
 6 legs  6 legs
 2 anal hooks
 Worm-like body
 Often build cases

Peckarsky, 1990
University of2009
Neuswanger, Wisconsin
Extension, 2007

Stonefly Nymph—Order Plecoptera
 6 legs
 Usually 2 tails
 Gills on thorax
 2 claws on end of each leg

Meyers, 2009
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Somewhat Sensitive:

Beetle Larvae—Order Coleoptera Marietta College, 2009
Neuswanger, 2009
 Abdomen composed of 8-10
Crayfish—Order Decapoda
segments
 Thoracic legs usually present
 2 large claws
 No terminal prolegs  8 legs
 2 long antennae

University of Minnesota, 2003

Clams—Class Pelecypoda Wisconsin Department of Natural Resources, 2008

 Two piece shell Damselfly/Dragonfly Nymph—Order Odonata
 Commonly oval
 Approximately 2-250 mm in size  Both have large labium (lower lip) and
moderately developed eyes
 No gills along the body
 Dragonfly larvae have 3 wedge-shaped
“tails” and the damselfly larvae have 3
feather-like “tails”

Clapp, 2006

Crane Fly Larva-Order Diptera
Dragonfly larva
 No wing pads Damselfly larva
 Worm-like body Clapp, 2006 Neuswanger, 2009
 8-10 abdominal segments
 May have prologs and terminal
processes
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South Dakota Game Fish and Parks, 2004

North Caroline Museum of Natural Sciences, 2005

Scud—Order Amphipoda Alderfly Larva—Order Megaloptera

 Laterally flattened  Abdomen with 7 pairs of 4-5
 Swims sideways segmented lateral filaments
 7 pairs of legs  A single unbranched terminal
 Resembles a shrimp filament

California Department of Fish and Game, 2009 Neuswanger, 2009

Sowbug—Order Isopoda

 7 pairs of legs and 2 antennae Net-spinning Caddisfly Larva—Order
 Dorsally flattened Trichoptera

 1-3 sclerotized (hardened) thoracic
segments
 3 pairs of legs
 branched gills may be present on
ventral side of abdomen
 anal hooks may also have tufts of
longer hair
Clapp, 2006

Fishfly Larva—Order Megaloptera

 No gill tufts underneath abdomen
 Resembles a small hellgrammite
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University of Minnesota, 2003

Tolerant:

Aquatic Worm-- Class Oligochaeta
(AECOS, 2002)
 Elongate cylindrical worms usually 1-
30 mm in length Midge Fly Larva—Order Diptera
 Anywhere from 7-500 body
segments  May have prolegs on thorax
 Terminal segment of abdomen may
have processes on it
 Worm-like body
 No wing pads
 May resemble caddisfly

Texas Flyfishers, 2006

Blackfly Larva—Order Diptera

 Cylindrical body with one end wider
 Head with fan-like mouth brushes

Neuswanger, 2009

Other Snails--Class Gastropoda
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Leech-- Order Hirudinea  Non-gill breathing snails
 Do not have an operculum
 Dorsoventrally flattened  Opening is usually on left side
 Anterior and posterior ventral
suckers
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(Environmental Protection Agency, 2009)

Quantifying Your Results:
On your data sheet, assign one of the following codes to each group of organisms:
R (rare) = 1 to 9 organisms
C (common) = 10 to 99 organisms
D (dominant) = 100 or more organisms

The data sheets separate the macroinvertebrates into pollution tolerance groups where:

Group I represents organisms that are very sensitive to pollution and tend to live only in good-
quality water

Group II represents organisms that are somewhat sensitive to pollution and are able to survive
in water quality that is either good or fair.

Group III represents organisms that are tolerant of pollution and are able to survive in good,
fair, and poor water quality

Once you have assigned the R, C, and D codes to the different groups, you are able to calculate
the index value as described on the data sheet. An example of this scoring is provided for you.
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EASI Tally Sheet (Example):
Group II
Group I Sensitive Count Somewhat Count Group III Tolerant Count
Sensitive
Water Penny Larvae 32 Beetle Larvae 0 Aquatic Worms 13

Hellgrammites 1 Clams 0 Blackfly Larvae 0

Mayfly Nymphs 46 Cranefly Larvae 0 Leeches 0

Crayfish 2

Gilled Snails 0 Damselfly Nymphs 1 Midge Larvae 3

Scuds 0

Riffle Beetles (adult) 0 Sowbugs 12 5
Snails

Stonefly Nymphs 7 Fishflies 0 Site Designation:

Alderflies 0

Non Net-Spinning Caddisfly Larvae 103 Net-Spinning 41 Team Members:
Caddisfly Larvae

Calculating the EASI Index Value to Rate the Water Quality of the Stream
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Macroinvertebrate Survey (Example):
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EASI Tally Sheet:
Group II
Group I Sensitive Count Somewhat Count Group III Tolerant Count
Sensitive
Water Penny Larvae Beetle Larvae Aquatic Worms

Hellgrammites Clams Blackfly Larvae

Mayfly Nymphs Cranefly Larvae Leeches

Crayfish

Gilled Snails Damselfly Nymphs Midge Larvae

Scuds

Riffle Beetles (adult) Sowbugs
Snails

Stonefly Nymphs Fishflies Site Designation:

Alderflies

Non Net-Spinning Caddisfly Larvae Net-Spinning Team Members:
Caddisfly Larvae
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Macroinvertebrate Survey:
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Glossary:
Aquatic Vegetation Beds—beds of submerged, green/leafy plants attached to stream bottom; typically
productive

Decaying Organic Matter- typically leaves or once-living organisms that are in the process of decaying

Reference site- a site on the stream which is unimpaired or minimally impaired compared to the other
sites being monitored

Riffle zone- an area of the stream that is shallower than the rest and has faster moving water, usually
broken up by rocks

Substrate—a material or substance on which an organisms grows and lives, for example rocks or soil

Snags and logs—submerged wood, usually dead trees, logs, branches, roots, and leaf packs; very
productive

Vegetated Bank Margin—consists of overhanging bank vegetation and submerged root mats attached
to banks as well as submerged, decomposing leaf packs; highly productive and most abundant type of
habitat.
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References:
AECOS. 2002. Invertebrate Keys Page. Retrieved April 20, 2009 from the CPIE Project.
Web site: http://www.aecos.com/CPIE/inv_02.html

California Department of Fish and Game. (2009). Aquatic Bioassessment Laboratory. Retrieved February
27, 2009.
Web site: http://www.dfg.ca.gov/abl/Lab/bug_tour/amphipoda.asp

Clapp, M. (2006). East Fork Lewis River Water Quality & Aquatic Macroinvertebrates Study. Retrieved
February 27, 2009, from Battle Ground Public School.
Web site: http://www.bgsd.k12.wa.us/hml/jr_cam/macros/amc/images/33.jpg

Environmental Protection Agency. 2009. Pouch Snails. Retrieved on April 20, 2009 from Biological
Indicators of Watershed Health.
Web site: http://www.epa.gov/bioindicators/html/pouchsnails.html

Lake County, Ohio. 2009. Macroinvertebrate images. Retrieved February 27, 2009, from Lake County of
Ohio.
Web site: http://www.lakecountyohio.org/soil/images-gif/Macroinvertebrates/Gill%20snail.gif

Marietta College. (2009). Index of wetlands. Retrieved February 27, 2009, from Department of Biology
and Environmental Science.
Web site: http://www.marietta.edu/~biol/biomes/images/wetlands/tipul2.jpg

Meyer, J. (2009). Entomology for Educators. Retrieved February 27, 2009, from North Carolina State
University.
Web site: http://www.cals.ncsu.edu/course/ent525/water/aquatic/images/15.jpg

Neuswanger, J. (2009). Aquatic Insects of American Trout Streams. Retrieved February 27, 2009, from
Troutnut. Web site: http://www.troutnut.com/im_regspec/picture_3659_small.jpg

North Carolina Museum of Natural Sciences (2003). Educator Trek. Retrieved February 27, 2009.
Web site: http://www.naturalsciences.org/education/treks/amphibian/images/Fishfly-larvae.jpg

Peckarsky,B. (1990). Freshwater Macroinvertebrates of Northeastern North America. Retrieved February
27, 2009.
Web site: http://www.swcd.co.trumbull.oh.us/

Senior Environmental Corps. Biosurvey In: Volunteer Water Quality Monitoring Field Manual.
Environmental Alliance for Senior Involvement.

South Dakota Game, Fish, and Parks. (2004). Wildlife Diversity Program. Retrieved 27, February, 2009.
Web site: http://www.sdgfp.info/Wildlife/Diversity/Digest%20Articles/caddis2.jpg
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Texas Flyfishers. (2006). Aquatic Worm. Retrieved February 27, 2009.
Web site: http://www.texasflyfishers.org/Aquatic%20Worm%20Annelid%20Guadalupe%20River.jpg

University of Minnesota. (2003). Volunteer Stream Monitoring Interactive Verification Program.
Retrieved February 27, 2009, from Chironomidae Research Group.
Web site: http://www.entomology.umn.edu/midge/WebKey/Philopotamidae.jpg

University of Wisconsin Extension. (2007). Level 1 Ecology. Retrieved February 27, 2009, from
Wisconsin’s Citizen-Based Water Monitoring Network.
Web site: http://watermonitoring.uwex.edu/images/level1/wav/ecology/Limnephilidae.jpg

Wisconsin Department of Natural Resources. (2008). Invasive Species. Retrieved February 27, 2009.
Web site: http://dnr.wi.gov/invasives/fact/images/RustyCrayfishJeffGunderson.jpg