1. KENTUCKY WATERSHED WATCH BIOLOGICAL STREAM ASSESSMENT High Gradient
Standard Operating Procedure
WWSOP03000
Effective Date: ##/##/####
This presentation has been developed from the information in the Watershed Watch in Kentucky (WWKY) Standard Operating Procedure (SOP) for the collection and analysis of macroinvertebrates from Kentucky high gradient streams.
Briefly explain that “gradient” refers to the slope of a stream and “high gradient” streams flow out of hills and mountains. Also inform volunteers that a more detailed explanation will come later in this presentation and in the habitat presentation.
In this module we will teach you how to correctly collect a macroinvertebrate sample for analysis to ensure accurate results that are representative of true stream conditions and comparable to other samples collected at our sites and at other sites across our watershed and the state. We will also give you an opportunity to practice what you have learned by demonstrating your newly acquired biological sample collection skills.

2. SOP statement This presentation is based on the
“Kentucky Watershed Watch High Gradient Biological Stream Assessment Standard Operating Procedure WWSOP03000”
Explain the information in this presentation was taken from the Kentucky Watershed Watch High Gradient Biological Stream Assessment Standard Operating Procedure (SOP) WWSOP03000 and encourage all volunteers to read this SOP to gain a deeper understanding of the information. The most current version of this SOP is available at the Watershed Watch website: water.ky.gov/wsw/Pages/default.aspx
The continued and expanded use of Watershed Watch data depends upon the training of volunteers in approved sampling procedures and the conscientious use of those procedures by each volunteer to ensure consistent, high quality sampling results across the state.

3. Be Safe!
•During high flow, sampling should be postponed until baseline conditions exist
•Waders and specialized wading boots should be utilized
•Use extreme caution when wading in streams above the knee
•Samplers should exhibit caution in and around streams to reduce the threat of a falling injury
•Secure permission from landowner
•Do not enter stream when combined air and water temperature is less than 120°F (to avoid frostbite or hypothermia)
•Do not stick hands or feet under large objects. Exercise caution when handling aquatic life. Some organisms pinch, bite, etc.
All water bodies have the potential to be contaminated
Notify others of your whereabouts and carry a cell phone
Use caution and be aware of your surroundings and location
Read through each bullet while emphasizing each volunteer’s personal responsibility for their safety and health.
Also explain that Watershed Watch expects each volunteer to put their personal safety above all else when participating in WW activities.

4. Introduction to Macroinvertebrates and Water Quality
Simplified scientific approach to stream water quality evaluation
Collection and analysis procedures used in this guide are designed to:
be low cost
be performed quickly and
easily
provide hands-on experience
for individuals, community
volunteer groups, and school
groups of all ages
Explain to the volunteers it is not the intent of this training and these procedures to pinpoint subtle changes in water quality, nor will it produce "research grade" information. However, it will produce solid scientific information that accurately represents the biological conditions in a stream and identify changes in the macroinvertebrates over time in response to changes in the watershed. It also doesn’t require extensive scientific training and experience or expensive laboratory equipment.

5. Streams are constantly changing
GRAB SAMPLE
Instantaneous water
quality
Quick process
Specific pollutant
analysis
BIOLOGICAL ASSESSMENT
Water quality over
time
Takes more time
Collective pollution
indication
Streams are the epitome of dynamic systems as they are constantly changing. The section of stream that is polluted today may be flushed clean next week. A grab sample collected at that time would not indicate that the pollution had occurred.
The effects of that pollution on the biological communities in the stream, however, would linger and may be dramatic, as reflected in changes in the variety and abundance of macroinvertebrate populations. A biological assessment provides a relative view of the overall quality of a stream over time.
VS.

6. Benthic Macroinvertebrates
Occurring at the bottom of a body of water

7. Benthic Macroinvertebrates
Visible to the naked eye
Freshwater macroinvertebrates are those organisms lacking a backbone that are visible to the naked eye, including insects, crustaceans (crayfish, etc.), mollusks (clams and mussels), gastropods (snails), oligochaetes (worms) and others.
Literally thousands of different species of macroinvertebrates have been found in Kentucky's streams and rivers.
Invertebrate
Lacking a backbone

8. Important!
Some mussels and other macroinvertebrates are Threatened or Endangered. Therefore, extra care must be taken to return all organisms to the stream.
In addition, any vertebrate organisms (ex. fish, snakes, etc.) must be returned to the stream as soon as you realize they have been captured.
Photo ID: left – mussel – Threeridge (Amblema plicata); right –Eastern Sand Darter (Ammocrypta pellucida)
Explain to volunteers it is illegal to harm any threatened or endangered species.

9. Macroinvertebrate Habitat
Macroinvertebrates have the same basic requirements as humans to thrive.
They need:
Oxygen to breathe
Food to eat
Shelter from
predators
Clean, pollution
free environment
Habitat - The place where a population (e.g. human, animal, plant, microorganism) lives and its surroundings, both living and non-living
Explain the relationship between a large variety of habitats in a stream that meet these basic needs and a healthy variety of macroinvertebrates found living there.

10. Tied to specific habitats Most are unable to avoid pollution
Macroinvertebrates are good indicators of the general health of streams for several reasons:
Easy to collect
Tied to specific habitats
Most are unable to avoid pollution
Different sensitivities and responses to pollution
Photo ID: stoneflies (Pteronarcys sp.) photo by Rich Merritt
They are commonly found in streams and are easy to collect
Their existence is tied to specific habitats within the stream system
For all or some portion of their life cycle, most are unable to move from these habitats or out of the stream to escape pollution or other disturbance
The various macroinvertebrate groups have different sensitivities and responses to pollution or disturbance in the stream system, so their presence or absence is a piece of information about the condition of the stream

11. Stream Types in Kentucky
High Gradient
Low Gradient
Overall steep slope
Originate in hilly terrain
More rapid flow
Many riffles
Overall low slope
Originate in flat terrain
Slower flow
No riffles
Creeks in Kentucky vary from high gradient with rocky bottoms and riffles to low gradient streams with sandy or silty bottoms and pools.
A high gradient indicates a steep slope and rapid flow of water, whereas a low gradient indicates a more nearly level stream bed and slowly moving water.
Low-gradient streams are defined as streams that have velocities less than 0.013m/sec (0.5ft/sec) and naturally lack riffle habitat.
Because of the differences in flow and depth regimes, stream bottom materials and habitats, oxygenation and temperature of water between the high and low gradient streams, the naturally occurring biological communities vary between the two.
Comparing the biological assessment or habitat assessment of a high gradient and a low gradient stream is comparing apples to oranges.
Because the biology and habitat are so different, there are different methods and measurements to use for high gradient and low gradient streams.

12. Instream Characteristics
Pools - The deepest areas with the slowest flow in a stream
Riffles - The areas in a stream where there is a drop in elevation that causes the
rocks on the bottom of the stream to extend above the surface of the water creating
a churning action that causes the surface of the water to look rough and bubbly
Runs - The segments of streams that flow quickly out of a riffle or into a pool
High Gradient
Instream Characteristics
Point the volunteers’ attention to the graphic on the slide and discuss the different depths, flow speeds, and surface appearance of each area of the high gradient stream

13. Instream Characteristics
Low Gradient
Instream Characteristics
Pools - The deepest areas with the slowest flow in a stream
Runs - The segments of streams between pools that are shallower and flow more quickly
Point the volunteers’ attention to the graphic on the slide and discuss the different depths, flow speeds, and surface appearance of each area of the low gradient stream

14. Single, identifiable source
Types of Pollution
Single, identifiable source
Point Source
Poor water quality may have a specific and observable source, such as a sewage treatment plant outfall or an industrial discharge. These are referred to as point sources of pollution.
However, not all sources of pollution will be easily identifiable. Most pollution is carried into streams by rain and snowmelt running off the ground surface. This is referred to as nonpoint source pollution.
Most nonpoint source pollution is from land that is:
built on or paved
farmed
bare soil
All types of pollution can have impacts on macroinvertebrates that live in streams.
Comes from entire watershed
Nonpoint Source

15. Stream Pollution and Disturbance
Photo ID: left – bacteria inputs, nutrient enrichment and trampling of stream banks by cows in creek; center – chemical pollution of Cuyahoga River in Ohio led to river fire in the 60’s; right – physical destruction of streambed habitat by heavy equipment in the stream
The introduction of human and livestock wastes and agricultural fertilizers results in bacteria and organic enrichment or pollution. Small quantities of these wastes or chemicals can cause organic enrichment resulting in excessive blooms of algae and other aquatic plants. As these plants die and fall to the stream bottom, they decompose and consume dissolved oxygen in the water. This can result in severe oxygen depletion, which eliminates the sensitive types of macroinvertebrates, leaving only those that can tolerate extremely low oxygen levels. Additionally, the dead plants on the stream bottom create a plentiful food supply that favors detritus feeders, which can lead to reductions in the diversity of other macroinvertebrate feeding groups.
Chemical pollutants such as chlorine, acids, metals, pesticides (including herbicides) and oil affect macroinvertebrates according to their tolerance to the pollutant. Generally speaking, however, a toxicity problem is usually the only condition that will render a stream totally devoid of macroinvertebrates.
Other stream conditions can cause variations in kinds and numbers of macroinvertebrates at a particular collection site. These conditions impact the variety of habitat needed to support diverse aquatic life. Examples of these conditions include stream alterations (such as straightening), siltation, and loss of streamside vegetation.

16. Pre-sampling Information and Procedures
The Importance of Habitat
Biological Assessment
and
Habitat Assessment
go hand in hand
To determine physical problems affecting the stream, a habitat assessment of each sampling site should be conducted at the same time as the biological assessment to determine the overall quality of the habitat for macroinvertebrates.
This includes the measurement and estimation of certain stream characteristics and the evaluation of the watershed for potential sources of pollution.
The results of this assessment will indicate whether the differences in numbers and kinds of macroinvertebrates may be due to natural variations in habitat characteristics or the result of human influence.
The biological assessment and habitat assessment should be done on the same day if possible. This helps to ensure that the two sets of information collected support each other (i.e. no major land use changes occur between events).
The biological sampling should be conducted first to avoid disturbing potential sampling locations. Additionally, having sampled the various microhabitats and walked the reach helps ensure a more accurate habitat assessment.
It is helpful to conduct the biological assessment and habitat assessment with another trained person, if possible. This provides you with a collaborator with whom you can discuss any questions about identifications or scores that may arise.

17. Reach Selection
A 100 meter reach that contains your grab sampling site and that is representative of the characteristics of the stream should be selected.
NOTE: Following periods of very high water, populations of organisms may be temporarily disrupted. Several weeks to a month may be required to reestablish the benthic community
Photo ID: a tape measure indicating the 100 meter reach distance
Reach - The selected length of a stream
Whenever possible, the area should be at least 100 meters upstream from any road or bridge crossing to minimize its effect on stream velocity, depth, and overall habitat quality. There should be no major tributaries discharging to the stream in the study area.

18. Reach Selection
The 100 meter measurement should be taken down the middle of the stream channel (following the path of the stream as closely as safety allows).
If it is too hazardous to measure down the middle of the channel, then move to the edge or along the adjacent stream bank.

19. Biological Assessments must be done in
Sample Scheduling
Biological Assessments must be done in
when macroinvertebrate groups are most easily found and before water levels drop in small streams
May or
June
Because many volunteers’ sites are in smaller, headwater streams, biological assessments in Kentucky must be done in the spring to ensure adequate water levels and appropriate representation of macroinvertebrate groups.
Assessments must be done in either the month of May or June.

20. Macroinvertebrate Collection Procedures
Professional macroinvertebrate samples
Professional macroinvertebrate samples must be taken in a very comprehensive manner for later identification to the genus or species level.
Each sample can require up to an hour to collect and process for storage, and several additional days for analysis using expensive laboratory equipment.
This process, while essential to providing data of scientific and legal quality, is costly and requires a high level of scientific expertise.

21. Macroinvertebrate Collection Procedures
Watershed Watch Biological Stream Assessment – simplified scientific methods
The Watershed Watch Biological Stream Assessment utilizes similar techniques, but has been developed using low-cost, simplified methods that provide a quicker means of sampling stream areas.
While streamlined, these assessment procedures use scientific methods to provide an accurate representation of screening-level stream conditions and can be accomplished by trained volunteers in a relatively short time period.

22. Enter downstream and work your way upstream
DON’T trudge through the area you will be sampling and dislodge everything you to sample
It is important not to disturb the in-stream habitat that you will be sampling before you have collected your samples.
Assessment procedures call for field identification with organisms returned to the stream.
To record stream and sampler information, use the “Kentucky Watershed Watch Biological Assessment Field Record” (Field Record).
Enter downstream and work your way upstream
so you won’t disturb the in-stream habitat you will be sampling

23. Basic equipment needed:
Photo ID: Clockwise from boots - waterproof boots(or waders-far right), two white plastic pans, first aid kit(optional), forceps, GPS unit(optional), D-frame dip net, latex/vinyl gloves(optional), Softnet dip net(no longer usable for this procedure), forms, clipboard(optional), pencil/indelible pen.
Other basic equipment needed but not in photo:
required - all Biological Assessment Field Guidance cards, thermometer
optional – bucket, hand lens, digital camera
The required equipment will be provided by Watershed Watch, but most of the optional equipment must be provided by the volunteer

24. High Gradient/Rocky Bottom Stream Sampling Procedure
Rocky bottoms (or hard substrate) will be prevalent in the riffles (and runs), which are a common feature throughout most mountain and piedmont streams.
Direct volunteers attention to the Field Guidance for Kentucky Watershed Watch High Gradient Biological Stream Assessment (WWSOP03002), which is a summary of the following slides. This will be made available to each volunteer as a portable document in the form of laminated cards on a ring for use as a reminder of the steps in the procedure while at the stream performing your assessment

25. Kick - accomplished by using toe and/or heel
SAMPLING TECHNIQUES:
Kick - accomplished by using toe and/or heel
of shoe to dislodge upper layer of cobble or gravel and scrape underlying bed, allowing downstream flow to carry organisms into net
The kick is conducted very vigorously with maximum movement of substrate and thorough dislodging of organisms as the goals.
Large rocks, leaves and sticks should be hand washed into the net.

26. SAMPLING TECHNIQUES:
Swish - accomplished by placing item in water in first collection pan and moving it vigorously through water several times to dislodge organisms
Place any organisms found into the second collection pan.
Avoid transferring debris to the second pan.

27. SAMPLING TECHNIQUES:
Jab - accomplished by thrusting D-frame net into targeted habitat in a jabbing motion for approximately 0.5 m (18 in.) and then sweeping water in jabbed area with net two or three times to collect the dislodged organisms

28. For each sampling technique used:
SAMPLING TECHNIQUES:
For each sampling technique used:
In each habitat type sampled, vigorously disturb
habitat until:
Habitat surface is thoroughly disturbed
No more materials are dislodged into your net
(For riffle) all materials on top of stream bed
have been disturbed in sample location
The sampling techniques will be described in the following slides.

29. SAMPLING TECHNIQUES:
For each technique in each habitat type sampled, use the same level of effort to perform the sample collection.
Emphasize the importance of using the same level of effort to ensure the macroinvertebrates collected are truly representative of the groups that are actually leaving in the stream.

30. What to do if you collect large quantities of
SAMPLING TECHNIQUES:
What to do if you collect large quantities of
small or fine material (such as sand or silt)?
To speed up the process of inspecting this material for macroinvertebrates:
Use a bucket and a rinsed net
Half-fill bucket with stream water
Place small handfuls of fine material into bucket
Vigorously swirl water in bucket to separate
macroinvertebrates from fine material
Immediately after swirling, pour water only (not fine
material) through net to capture any suspended
macroinvertebrates and place them in first pan
Pour the water in an area away from your collection pans
Repeat until all of fine material has been processed
Instructors should demonstrate the swirling technique so it is clearly understood
This technique can be used for any sample in which the net becomes clogged

31. The following slides describe the possible
HABITAT TYPES:
The following slides describe the possible
Habitat Types to be sampled and identify
Techniques to be used for each:

32. Riffles
Root Mat Definition - A side of the stream channel where the soil has been eroded, creating an overhang of soil (undercut bank) that may or may not be covered with vegetation. If vegetation is present on the overhang, the roots may extend down through the soil and hang freely in mats below the undercut bank in contact with the stream.
Undercut banks without root mats are also sampled
Types of macroinvertebrates found in the root mat habitat include: dragonflies and damselflies

33. Kick a 0.25m2 (18 in. x 18 in.) area upstream of net
Riffles
Place net on substrate in middle area of riffle where water is flowing fastest
Kick a 0.25m2 (18 in. x 18 in.) area upstream of net
Large rocks, leaves and sticks should be hand washed into net
Root Mat Definition - A side of the stream channel where the soil has been eroded, creating an overhang of soil (undercut bank) that may or may not be covered with vegetation. If vegetation is present on the overhang, the roots may extend down through the soil and hang freely in mats below the undercut bank in contact with the stream.
Undercut banks without root mats are also sampled
Types of macroinvertebrates found in the root mat habitat include: dragonflies and damselflies

34. Root Mats Associated with Undercut Banks
Root Mat Definition - A side of the stream channel where the soil has been eroded, creating an overhang of soil (undercut bank) that may or may not be covered with vegetation. If vegetation is present on the overhang, the roots may extend down through the soil and hang freely in mats below the undercut bank in contact with the stream.
Undercut banks without root mats are also sampled
Types of macroinvertebrates found in the root mat habitat include: dragonflies and damselflies

35. Root Mats Associated with Undercut Banks
Place large root wad into D-frame dip net and shake vigorously
For undercut banks without root mats, use jab
Root Mat Definition - A side of the stream channel where the soil has been eroded, creating an overhang of soil (undercut bank) that may or may not be covered with vegetation. If vegetation is present on the overhang, the roots may extend down through the soil and hang freely in mats below the undercut bank in contact with the stream.
Undercut banks without root mats are also sampled
Types of macroinvertebrates found in the root mat habitat include: dragonflies and damselflies

36. Aquatic Vegetation (Submerged and Emergent)
Aquatic Vegetation Definition - Aquatic plants located above and/or below the stream surface, including masses of algae; may be rooted in the stream bottom or free-floating
Types of macroinvertebrates found in the aquatic vegetation habitat include: dragonflies and damselflies

37. Aquatic Vegetation (Submerged and Emergent)
Use modified jab, thrusting dip net into vegetation for approximately 1 meter (jab below water surface from bottom to top of
vegetation) and
sweeping through
area to collect
dislodged organisms
Aquatic Vegetation Definition - Aquatic plants located above and/or below the stream surface, including masses of algae; may be rooted in the stream bottom or free-floating
Types of macroinvertebrates found in the aquatic vegetation habitat include: dragonflies and damselflies

38. Bedrock or Slab-Rock
Bedrock or Slab-Rock Definition - Stream bottom that is a solid layer of rock
The reduced variety of types of spaces for macroinvertebrates to live in on a bedrock/slab-rock bottom stream result in reduced variety/numbers of macroinvertebrates found living there
Types of macroinvertebrates found in the bedrock/slabrock habitat include: mayflies, caddisflies and stoneflies

39. Place edge of dip net flush on substrate with opening facing upstream
Bedrock or Slab-Rock
Place edge of dip net flush on substrate with opening facing upstream
Kick approximately
0.1 m2 (11 in. x 11 in.)
of area immediately
upstream of net
Bedrock or Slab-Rock Definition - Stream bottom that is a solid layer of rock
The reduced variety of types of spaces for macroinvertebrates to live in on a bedrock/slab-rock bottom stream result in reduced variety/numbers of macroinvertebrates found living there
Types of macroinvertebrates found in the bedrock/slabrock habitat include: mayflies, caddisflies and stoneflies

40. Leaf Packs
Leaf Pack Definition - Clumps of leaves and/or small sticks that have floated downstream and become trapped by rocks, root wads, woody debris, etc.
These leaf packs must be made up mostly of leaves that are conditioned (that have been in the stream for a while and have begun to break down). This ensures two things:
the leaf material is more easily eaten and digested
the macroinvertebrates have had time to find and use the leaf pack
Types of macroinvertebrates found in the leaf pack habitat include: mayflies, caddisflies and stoneflies

41. Take a large handful from each type of area
Leaf Packs
Take a large handful from each type of area
present (i.e., riffles, runs
and pools) and place
into net
NOTE: Collect from
“conditioned”
(i.e., beginning to
break down) material
Leaf Pack Definition - Clumps of leaves and/or small sticks that have floated downstream and become trapped by rocks, root wads, woody debris, etc.
These leaf packs must be made up mostly of leaves that are conditioned (that have been in the stream for a while and have begun to break down). This ensures two things:
the leaf material is more easily eaten and digested
the macroinvertebrates have had time to find and use the leaf pack
Types of macroinvertebrates found in the leaf pack habitat include: mayflies, caddisflies and stoneflies

42. Large Submerged Wood
Large Submerged Wood Definition - Pieces of submerged wood, ranging from roughly 3 to 6 meters (10 to 20 linear feet) and ranging from 5–15 cm (2–6 inches) in diameter
These large pieces of submerged wood must be in the stream for a while and have begun to break down. This ensures two things:
the wood material is more easily eaten and digested
the macroinvertebrates have had time to find and colonize the wood
Types of macroinvertebrates found in the large submerged wood habitat include: beetle larvae

43. Large Submerged Wood
Jab pieces of submerged wood, ranging from roughly 3 to 6 meters in length (10 to 20 linear feet) and ranging from 5–15 cm (2–6 inches) in diameter
Large Submerged Wood Definition - Pieces of submerged wood, ranging from roughly 3 to 6 meters (10 to 20 linear feet) and ranging from 5–15 cm (2–6 inches) in diameter
These large pieces of submerged wood must be in the stream for a while and have begun to break down. This ensures two things:
the wood material is more easily eaten and digested
the macroinvertebrates have had time to find and colonize the wood
Types of macroinvertebrates found in the large submerged wood habitat include: beetle larvae

44. Silt, Sand, and Fine Gravel (Submerged)
Silt, Sand and Fine Gravel Definition - Substrate consisting of sediment with particle sizes that are larger than clay (which will form ribbons when rubbed between thumb and fingers), and smaller than a BB (ammunition for a BB gun), approximately 4mm in diameter
Substrate - The material that forms the bed, or bottom, of a water body
Clarify to the volunteers the deposits to be sampled are loose, unpacked sediments beneath the surface of the water. Hard, packed deposits aren’t habitats where macroinvertebrates can easily live. Also, deposits that extend above the surface of the water are not habitable by aquatic macroinvertebrates either.
Types of macroinvertebrates found in the silt, sand, and fine gravel habitats include: burrowing mayflies and blood worms

45. Silt, Sand, and Fine Gravel (Submerged)
Sample this habitat last
Scoop a double handful of substrate by spreading hands 1 foot apart and bringing both hands together in a sweeping motion at a depth of 2 to 3 inches into the substrate
Place material into first collection
pan and work through with fingers
to inspect (or use Bucket method)
NOTE: This substrate consists of
sediment with particle sizes that
are larger than clay and smaller
than a BB
Scooping (is defined on this slide) – spread hands 1 foot apart and bring both hands together in a sweeping motion to collect a double handful of the substrate
Silt, Sand and Fine Gravel Definition - Substrate consisting of sediment with particle sizes that are larger than clay (which will form ribbons when rubbed between thumb and fingers), and smaller than a BB (ammunition for a BB gun), approximately 4mm in diameter
Substrate - The material that forms the bed, or bottom, of a water body
Sample this habitat last because these materials can cover other materials or organisms, obscuring them from view
Clarify to the volunteers the deposits to be sampled are loose, unpacked sediments beneath the surface of the water. Hard, packed deposits aren’t habitats where macroinvertebrates can easily live. Also, deposits that extend above the surface of the water are not habitable by aquatic macroinvertebrates either.
Types of macroinvertebrates found in the silt, sand, and fine gravel habitats include: burrowing mayflies and blood worms

46. representative of stream characteristics
SAMPLING PROCEDURES:
The first time you conduct this assessment, select and measure a 100 meter reach containing your grab sampling site and
representative of stream
characteristics
Return to this same reach for all
future assessments
Sampling begins at downstream
end of reach and proceeds
upstream
It is important to sample all habitat types that exist within the selected 100 meter reach.
A composite sample is taken by combining organisms from individual sampling spots in the riffles and other areas into one collection pan.
Emphasize that 100 meters is a long distance and should not be estimated but actually measured with a tape, or marked string, on the initial visit to your site. In experiments with volunteers estimating a 100 m distance, they almost always underestimate the distance.

47. SAMPLING PROCEDURES:
Fill first collection pan at least half full of stream water (if it is too full, organisms may slosh out)
Place a small amount of water in the second collection pan

48. locations (divide among various riffles in reach)
SAMPLING PROCEDURES:
Kick in four riffle
locations (divide among various riffles in reach)
Sample once in each of other Habitat Types,
if present
Using a D-frame net, perform kick sampling in four locations from various riffles throughout the 100-meter reach.
Place your net on the substrate in the middle area of the riffle where the water is flowing the fastest.
A 0.25 m2 (18 in. x 18 in.) area upstream of the net should be vigorously disturbed using the kick.

49. SAMPLING PROCEDURES:
After sampling each location, transfer any collected material from net (or hands for sand, silt, and fine gravel) to the first collection pan –
invert net to transfer
Swish inverted net and
inspect for organisms. Forceps
may be needed to remove
organisms from net. Place any
organisms found into second
collection pan
NOTE: Samples from all individual sampling spots are combined to make one composite sample for reach .
Forceps may be needed to remove organisms from the net.
Place any organisms found into the second collection pan.
Avoid transferring debris to the second pan.

50. SAMPLING PROCEDURES:
Remove large debris from first collection pan, swish and discard after inspecting debris for organisms
Many of these organisms may
be quite small – take time to
swish and thoroughly inspect the materials before discarding
Pick any macroinvertebrates from first collection pan into second collection pan – avoid transferring debris

51. If first collection pan becomes full or muddy:
SAMPLING PROCEDURES:
If first collection pan becomes full or muddy:
Transfer all macroinvertebrates collected to that point into second collection pan
After all macroinvertebrates are
removed from first collection pan,
discard water and replace with new
water from stream
Continue sampling and placing
new material collected in first pan
until reaching required number of samples
Repeat as needed .
Forceps may be needed to remove organisms from the net.
Place any organisms found into the second collection pan.
Avoid transferring debris to the second pan.

52. Count only living organisms (empty shells should not be counted)
SAMPLING PROCEDURES:
Inspect first collection pan to ensure all organisms have been transferred to second collection pan
Using “Kentucky Water Watch Benthic Macroinvertebrate Identification Key”, identify and count organisms in second pan
Count only living organisms (empty shells should
not be counted)
Organisms that have been freshly killed by the collection procedure can be counted.

53. As you identify organisms
SAMPLING PROCEDURES:
As you identify organisms
in second collection pan, record your count on “Kentucky Watershed Watch Biological Assessment Field Data Sheet” (Field Data Sheet)
Emphasize to class that images on sheet and in Macroinvertebrate Key are not to scale. On their first biological assessment trip to the stream, volunteers often expect to see macroinvertebrates the size of the pictures they are given.
Use the “Kentucky Water Watch Benthic Macroinvertebrate Identification Key” (Macroinvertebrate Key) to assist you with identification if needed.
Record the Abundance Count (number of organisms) in the boxes to the right of any organisms collected.
You can stop counting when you reach 30 (write “30” in the box) for any one type of organism. However, if you do count 30 for any one type of organism, please estimate if the count would be more than 50 organisms. If so, write “50+” in the box.

54. Macroinvertebrate Identification
Instruct volunteers to get out the Macroinvertebrate Key they were given in their kit.
Explain that this will be a practice demonstration for identifying a macroinvertebrate they might collect at their stream site.
Point out the identifying features on the slides after the correct answers appear.
Again emphasize the slides and photos are not to scale. They are very enlarged for instructional purposes and most macroinvertebrates collected will be small to very small and a hand lens may be needed to see the features being described in the Macroinvertebrate Key.

55. Macroinvertebrate Identification
Read through the three options on page 1 of the key while this slide is being displayed.
A red circle around the correct answer will appear when you hit enter after all students have had time to decide on their choice.
Explain the #7 after the correct answer indicates the number of the next card they turn to.

56. Macroinvertebrate Identification
Read the options on #7 and ask the volunteers to choose the one they think is correct while this slide is displayed.
A red circle around the correct answer will appear when you hit enter after all students have had time to decide on their choice.

57. Macroinvertebrate Identification
Read the options on #8 and ask the volunteers to choose the one they think is correct while this slide is displayed.
A red circle around the correct answer will appear when you hit enter after all students have had time to decide on their choice.

58. Macroinvertebrate Identification
Confirm this is a photo of a mayfly and instruct the volunteers to turn to #28 for further information on mayflies.

59. Macroinvertebrate Identification
Discuss the facts about mayflies on #28.
Emphasize the variations in appearance (color, size, shape, etc.) among different species of mayflies and explain there are many more than the three pictured on #28.
Pass the mayfly specimen from the provided macroinvertebrate kit around the room, so everyone can see what they potentially will look like.

60. Macroinvertebrate Identification
STOP! DO NOT advance this slide until volunteers have the opportunity to key out this photo on their own.
Instruct volunteers to now attempt to identify this macroinvertebrate on their own using the Macroinvertebrate Key and the previous process.
Allow reasonable time for most, if not all, in the class to identify it before advancing to the next slide with the answers.
Use key to identify on your own

61. Macroinvertebrate Identification
Reveal the correct answer on this slide and advance to next slide to go through the correct answers on the Macroinvertebrate Key that lead to #19.

62. Macroinvertebrate Identification
Go through the correct answers on the Macroinvertebrate Key that lead to #19.
Discuss the facts about true flies-blackflies on #19.
Pass the blackfly specimen from the provided macroinvertebrate kit around the room, so everyone can see what they potentially will look like.

63. Return organisms to stream as soon as Field Data Sheet is completed
SAMPLING PROCEDURES:
Return organisms to stream as soon as
Field Data Sheet is completed
Photo ID: clockwise from far left: crayfish, stonefly, damselfly, mayfly, non-operculate snail, midges, leech, watersnipe, caddisfly
Explain that high-tolerance species may still be present, but that isn’t a bad thing if they are balanced by the presence of low-tolerance species.
Example: the presence of leeches in the varied group of collected macroinvertebrates represented on this slide doesn’t indicate a problem in the stream. Leeches are an important part of a healthy biological community. If the only macroinvertebrates collected were leeches, this would be an indication of a problem in the stream.
Diversity is the key.
Also, explain that natural systems are variable in time and space, so we can’t simply set a critical number of species that must be present to classify a stream as healthy.

64. SAMPLING PROCEDURES:
Record each habitat type sampled in reach and any related comments on the “Kentucky Watershed Watch Biological Assessment Field Record” (Field Record)
NOTE: Percent of Reach is not required for high gradient streams
Tell volunteers to:
Note the type of net used for sampling
Comment on anything that would indicate adverse sampling conditions, e.g., high flows, treacherous rocks, difficult access to stream
Be aware the “*Percent of Reach” section is only used by those who are doing assessments in low gradient streams

65. SAMPLING PROCEDURES:
Place thermometer at mid-depth of stream in location with constantly flowing water
Leave thermometer there until reading stabilizes
Record stream temperature in degrees Celsius (ºC) on your Field Record
Tell volunteers to:
Record stream temperature in degrees Celsius
Use same sampling method used for Phase 1 temperature collection

66. POST-SAMPLING PROCEDURES:
Use the “Kentucky Watershed Watch Biological Assessment Calculation Sheet” (Calculation Sheet) to determine the biological Integrity Rating for your site
Instructions on how to complete this sheet can be found one following slides and in the KENTUCKY WATERSHED WATCH HIGH GRADIENT BIOLOGICAL STREAM ASSESSMENT Standard Operating Procedure WWSOP03000.
This step does not have to be completed at the stream.

67. Instructions – “Kentucky Watershed Watch Biological Assessment Calculation Sheet” (Calculation Sheet):
Transfer all Abundance Count numbers from the Field Data Sheet to the column labeled “Abundance Count”
The electronic form (Excel spreadsheet) will auto-calculate your Biotic Index number based on these Abundance Count numbers.

68. Calculation Sheet Instructions
Transfer the Biotic Index number to the box labeled “Biotic Index” on your Field Record

69. Calculation Sheet Instructions
Find the Bioregion for your site in the “Bioregion” box located on your Field Record

70. Calculation Sheet Instructions
Once your Bioregion is determined, use the Biotic Index number and the “Integrity Rating Chart” to determine the Integrity Rating for your site and record in the box labeled “Integrity Rating”

71. POST-SAMPLING PROCEDURES:
After biological sampling has been completed, use the “Kentucky Watershed Watch High Gradient Stream Habitat Assessment (WWSOP04000)” and “Field Guidance for Kentucky Watershed Watch High Gradient Stream Habitat Assessment (WWSOP04002)” to complete the habitat assessment
Conduct habitat assessment with another trained person, if possible
Again emphasize the importance of:
The biological assessment and habitat assessment should be done on the same day if possible. This helps to ensure that the two sets of information collected support each other (i.e. no major land use changes occur between events).
The biological sampling should be conducted first to avoid disturbing potential sampling locations. Additionally, having sampled the various microhabitats and walked the reach helps ensure a more accurate habitat assessment.
It is helpful to conduct the biological assessment and habitat assessment with another trained person, if possible. This provides you with a collaborator with whom you can discuss any questions about identifications or scores that may arise.

72. FORM SUBMITTAL
Following each Habitat Assessment event, please mail a copy of all completed forms to:
KY Water Watch Office
200 Fair Oaks Lane, 4th Floor
Frankfort, KY 40601
Here the forms will be recorded in the KY Water Watch database and filed for safekeeping
If you have any questions or difficulties, please contact the KY Water Watch office at:
Talk about the importance of form submittal for good recordkeeping and let volunteers this is the best way to ensure that they will be able to access their data in the future

73. INTERPRETATION
The Watershed Watch Biological Stream Assessment analyzes the abundance and pollution tolerance of all macroinvertebrate groups collected to make conclusions about the health of the aquatic system.
The Watershed Watch assessment procedures use scientific methods to provide an accurate representation of screening-level stream conditions.
These procedures use the standard 100 meter reach as a scientific representation of the stream system and larger watershed conditions.

74. POLLUTION TOLERANCE
Different macroinvertebrate groups have different sensitivities and responses to pollution. These are referred to as pollution tolerance.
Tolerant
Explain a pollution tolerant group is one that can withstand higher levels of pollution.
Examples of very pollution tolerant groups include aquatic worms and leeches.
Explain conversely, groups that cannot survive in the presence of lower levels of pollution are considered pollution intolerant.
Mayflies and stoneflies are examples of very pollution intolerant groups.
Intolerant

75. ABUNDANCE
The number of organisms from a macroinvertebrate group present in the stream is referred to as abundance.
Abundance of Aquatic Worms and other tolerant organisms indicate organic pollution
Photo ID: aquatic worms and bloodworms in stream below wastewater treatment plant discharge point
Explain that as abundance of pollution tolerant groups increases, and pollution intolerant groups decreases, you can infer that there is some type(s) of pollution or disturbance present in the stream system.
For example, a collection that contains a high abundance of aquatic worms and leeches, as well as a low abundance or absence of mayflies and stoneflies, indicates the presence of pollution or disturbance.

76. ABUNDANCE VALUE Abundance Count = 32 (abundant) 8 (common) 1 (rare)
On your Calculation Sheet, abundance counts were transferred and abundance values were assigned. These individual abundance values are used as general representations of abundance of the organisms in the stream, and are used in the formula that determines overall stream health.
The three different abundance values are assigned to categorize the occurrence of each macroinvertebrate group into:
6=abundant, 3=common, 1=rare
Abundance
Value =
(abundant) 6
(common) 3
(rare) 1

77. TOLERANCE VALUE Stonefly = 2 Leech = 9 (Tolerant) (Intolerant)
Individual tolerance values are pre-assigned for each macroinvertebrate group on the Calculation Sheet. These values are specific to Kentucky, and have been determined through historic collections made by KDOW professional biologists.
Using a scale of zero to ten, these tolerance values are assigned based on each group’s average pollution tolerance, with ten being the most pollution tolerant.
By using these values (abundance and tolerance) to perform the calculations on the Calculation Sheet, you obtain a biotic index that is specific to your stream.
(Tolerant)
(Intolerant)

78. BIOREGIONS
The different areas resulting from the influences of Kentucky’s geology are called bioregions. The four bioregions are the:
Mountains (MT)
Bluegrass (BG)
Pennyroyal (PR)
Mississippi Valley/Interior River Lowland (MVIR)
Kentucky is divided into different geologic regions, which have different influences on the types of plants and animals present in and around streams that exist in those regions. The different areas resulting from these influences are called bioregions.
The four bioregions of Kentucky are the Mountains (MT), the Bluegrass (BG), the Pennyroyal (PR) and the Mississippi Valley/Interior River Lowland (MVIR).
By determining the bioregion in which your site is located, you can use the biotic index and the integrity rating chart to determine the integrity rating (or overall stream health) for your site.
Explain the importance of comparing the Biotic Index you calculated for your site to the correct bioregion scale on the Integrity Rating Chart. The scales on the Chart are adjusted for the variances in the actual biological communities found in the different Bioregions.
Example: If your biological sample is collected from your site in the Mountains and you calculate your Biotic Index to be 3.7, but you accidentally compare it to the scale on the Integrity Rating Chart for the Bluegrass, you will interpret the Integrity Rating for your stream as Good. When, in reality, based on the scale for the Mountains, it is only Fair.
There is an area of overlap of the Mississippi Valley/Interior River Lowland and Pennyroyal bioregions. This overlap occurs in the general area of the Land Between the Lakes region. This is not something that needs to be discussed with volunteers, unless they ask.
All existing sites in the overlap area will be assigned a bioregion based on determinations by the KDOW Water Quality Branch biologists and pre-populated in the Bioregion box on the Field Record.
All new sites that are entered into the WWKY database will automatically be assigned a bioregion based on a GIS layer provided by the Water Quality Branch. These will also be pre-populated in the Bioregion box on the Field Record.

79. INTERPRETING DATA STATEWIDE
This map shows the distribution of values for these assessments across Kentucky. Higher scores, in blue, indicate more diverse biological communities.
This is an example of what can be done with data when we use standard methods for statewide collection

80. Recertification Training: Phase 2 (Biological and Habitat Assessment)
Your sampling and assessment skills are considered
current after completing WWKY-approved training
You will be notified that training updates are required
if modifications are made to WWKY methods
Inform the volunteers the updates and certification for sampling help us as an organization stay current with the latest protocols and sampling techniques.

81. IMPORTANT: If you observe an outfall, pipe, or anything that might indicate illegal pollution of a waterway, contact KDOW to report complaints or concerns at during business hours.
For environmental emergencies such as spills of gas, oil or other substances, contact the Environmental Response Team at or
The only time you should contact the Environmental Response Team is for an environmental EMERGENCY. This can be equated to the environmental version of “911”.