SOP #EH-04

Benthic Macroinvertebrate Sampling & Processing

(Adapted from ERT/REAC SOP and 

Great Lakes National Program Office Sampling Method LG406 Revision 07)
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

TABLE OF CONTENTS

1.0 PURPOSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 1

2.0 RESPONSIBILITIES ............................................... Page 1

3.0 EQUIPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 1

4.0 METHOD SUMMARY .............................................. Page 2

5.0 SAMPLING PROCEDURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 3

5.1 Habitat Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 3

5.2 Sample Collection of Community Samples . . . . . . . . . . . . . . . . . . . . . . . . . Page 4

5.2.1 General Information ..................................... Page 4

5.2.2 Non-Wadable Waterbodies ............................... Page 4

5.2.3 Wadable Waterbodies ................................... Page 5

5.3 Sample Collection for Analytical Measurements .................... Page 6

5.4 Habitat Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 6

6.0 SAMPLE CONTAINERS AND LABELING ............................. Page 7

7.0 LABORATORY PROCESSING FOR COMMUNITY SAMPLES . . . . . . . . . . . . Page 7

7.1 Laboratory Equipment/Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 7

7.2 Subsampling and Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 7

7.3 Identification of Macroinvertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 9

8.0 SUBSAMPLE PROCEDURE MODIFICATIONS . . . . . . . . . . . . . . . . . . . . . . . . Page 10

9.0 DECONTAMINATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 11

10.0 RECORD KEEPING AND QUALITY CONTROL ...................... Page 11

10.1 Required Information ......................................... Page 11

10.2 Field Quality Control Samples ................................. Page 12

11.0 REFERENCES ................................................... Page 12
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

ATTACHMENT 1 Benthic Macroinvertebrate Field Data Sheet 

Physical Characterization/Water Quality Field Data Sheet

Habitat Assessment Field Data Sheet 

ATTACHMENT 2 Sample Log-in Sheet

Benthic Macroinvertebrate Laboratory Bench Sheet
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 1

SYNOPSIS: A standardized method for collecting benthic macroinvertebrates for

ecological assessment. This procedure is based on USEPA Rapid Bioassessment Protocol

III. Protocols for sample collection and handling are provided. 

1.0 PURPOSE

The purpose of this Standard Operating Procedure (SOP) is to provide a standardized method for

sampling the benthic population at hazardous waste sites. This protocol summarizes the USEPA

Rapid Bioassessment Protocol III (RBP III) for benthic macroinvertebrates. RBP III utilizes the

systematic field collection and analysis of major benthic taxa, and can detect subtle degrees of

impairment at potentially contaminated sites. Discrimination of four levels of impairment should

be possible with this assessment. This SOP may be used by employees of USEPA Region 8, or

contractors and subcontractors supporting USEPA Region 8 projects and tasks. Deviations from

the procedures outlined in this document must be approved by the USEPA Region 8 Remedial

Project Manager, Regional Toxicologist or On-Scene Coordinator prior to initiation of the

sampling activity.

2.0 RESPONSIBILITIES

The Field Project Leader (FPL) may be an USEPA employee or contractor who is responsible

for overseeing the benthic macroinvertebrate sampling activities. The FPL is also responsible for

checking all work performed and verifying that the work satisfies the specific tasks outlined by

this SOP and the Project Plan. It is the responsibility of the FPL to communicate with the Field

Personnel regarding specific collection objectives and anticipated situations that require any

deviation from the Project Plan. It is also the responsibility of the FPL to communicate the need

for any deviations from the Project Plan with the appropriate USEPA Region 8 personnel

(Remedial Project Manager, Regional Toxicologist or On-Scene Coordinator).

Field personnel performing benthic macroinvertebrate sampling are responsible for adhering to

the applicable tasks outlined in this procedure while collecting samples.

3.0 EQUIPMENT

• D-frame dip net - 0.3 m2

 "D"-shaped net (500um nytex screen) where the net attaches to a

long pole. Net is cone-shaped for capture of organisms.

• Kick-net - 1 m2 net (500um nytex screen) attached to 2 poles, which functions in a

similar manner to a fish kick seine. 
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 2

• Ponar/Ekman Dredge - for collection of sediment-dwelling invertebrates in non-wadable

waterbodies.

• Elutriator Bucket or 500 um Mesh Sieve Bucket - used to remove invertebrates from

sediment.

• Collection containers - wide-mouth bottles (500 to 1,000 ml capacity).

• Gloves - for personal protection and to prevent cross-contamination of samples. May be

plastic or latex; should be disposable and powderless.

• Field notebook - a bound book used to record progress of sampling effort and record any

problems and field observations during sampling.

• Three-ring binder book- to store necessary forms used to record and track samples

collected at the site. Binders will contain the Benthic Macroinvertebrate Field Data

Sheet, Physical Characterization/Water Quality Field Data Sheet, and sample labels. 

Example forms are provided in Attachment 1.

• Permanent marking pen - used to label samples and to record information in field

logbooks and data sheets.

• Sieve Buckets - with 500um mesh. Must have 10 - 12 liter capacity.

• Forceps - to pick organisms from mesh screens and collection nets.

• 95% Ethanol - to preserve samples for analysis.

• Trash Bag - used to dispose of gloves and any other non-hazardous waste generated

during sampling.

4.0 METHOD SUMMARY

Benthic macroinvertebrates are collected systematically from all available in-stream habitats by

kicking the substrate or jabbing with a D-frame dip net. A total of 20 jabs (or kicks) are taken

from all major habitat types in the reach, resulting in sampling approximately 3.1 m2

 of habitat. 

An organism-based subsample (usually 100, 200, 300, or 500 organisms) is sorted in the

laboratory and identified to the lowest practical taxon, generally genus or species.
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 3

5.0 SAMPLING PROCEDURES

A 100m reach that is representative of the characteristics of the stream should be selected. 

Whenever possible, the area should be at least 100m 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. If a 100m reach is not available for

sampling, a standard number of stream widths can be used to measure the stream distance. For

example, the EPA's Environmental Monitoring and Assessment Program (EMAP) uses a

standard of 40 stream widths for sampling. This approach allows variation in the length of the

reach, based on the size of the stream.

Before sampling, complete the physical/chemical field sheet to document site description,

weather conditions, and land use. Example forms are provided in Attachment 1. After

sampling, review this information for accuracy and completeness.

Draw a map of the sampling reach or stream widths on the Field Data Sheet. This map should

include in-stream attributes (e.g., riffles, falls, fallen trees, pools, bends, etc.) and important

structures, plants, and attributes of the bank and near stream areas. Use an arrow to indicate the

direction of flow. Indicate the areas that were sampled for macroinvertebrates, with each sample

identification number on the map. Use a hand-held GPS for latitude and longitude determination

of the furthest upstream and downstream points of the sampling reach.

5.1 Habitat Types

The following major stream habitat types are colonized by macroinvertebrates and generally

support macroinvertebrate diversity in stream ecosystems. Some combination of these habitats

will be sampled using this multi-habitat approach to benthic sampling.

Cobble (hard substrate) - In many high-gradient streams, this habitat type will be dominant. 

Sample shallow areas with coarse (mixed gravel, cobble or larger) substrates by holding the

bottom of the dip net against the substrate and dislodging organisms by kicking the substrate for

0.5 m upstream of the net.

Snags - Snags and other woody debris that have been submerged for a relatively long period (not

recent deadfall) provide excellent colonization habitat. Sample submerged woody debris by

jabbing in medium-sized snag material (sticks and branches). The snag habitat may be kicked

first to help dislodge organisms, but only after placing the net downstream of the snag. 

Accumulated woody material in pool areas are considered snag habitat. Large logs should be

avoided because they are generally difficult to sample adequately.
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 4

Vegetated banks - When lower banks are submerged and have roots and emergent plants

associated with them, they are sampled in a fashion similar to snags. Submerged areas of

undercut banks are good habitats to sample. Sample banks with protruding roots and plants by

jabbing into the habitat. Bank habitat can be kicked first to help dislodge organisms, but only

after placing the net downstream. 

Submerged macrophytes - Submerged macrophytes are seasonal in their occurrence and may not

be a common feature of many streams, particularly those that are high-gradient. Sample aquatic

plants that are rooted on the bottom of the stream in deep water by drawing the net through the

vegetation from the bottom to the surface of the water (maximum of 0.5m each jab). In shallow

water, sample by bumping or jabbing the net along the bottom in the rooted area, avoiding

sediments where possible.

Sand (and other fine sediment) - Usually the least productive macroinvertebrate habitat in

streams, this habitat may be the most prevalent in some streams. Sample banks of unvegetated

or soft soil by bumping the net along the surface of the substrate rather than dragging the net

through soft substrates; this reduces the amount of debris in the sample. 

5.2 Sample Collection of Community Samples

5.2.1 General Information

Record the percentage of each habitat type in the reach. Note the sampling gear used, and

comment on conditions of the sampling, e.g., high flows, treacherous rocks, difficult access to

stream, or anything that would indicate adverse sampling conditions.

Document observations of aquatic flora and fauna. Make qualitative estimates of

macroinvertebrate composition and relative abundance as a cursory estimate of ecosystem health

and to check adequacy of sampling.

5.2.2 Non-Wadable Waterbodies

Non-wadable waterbodies should use a Ponar or Ekman dredge to collect sediment-dwelling

benthic macroinvertebrates. Sediment collection procedures are similar to the collection

techniques discussed in the Sediment Sampling SOP (#EH-03). Multiple sediment grab samples

will be composited and placed in an elutriator bucket or 500um mesh sieve bucket. Add water to

the sample and hand-mix gently to break up lumps of sediment. Pour the sample slurry from the

tub through the elutriator or sieve bucket which is placed over a second tub to catch the rinse

water. Wash the sediment through the mesh with water at very low pressure. Excessive pressure

will result in damage to organisms, in particular oligochaetes, and could compromise taxonomic
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 5

analysis of the sample. Gently agitate the sieve bucket to aid in rinsing the fine sediment out of

the sample. It may be necessary to sieve the slurry in small portions to prevent clogging of the

mesh. Continue to rinse the composite with surface water until all sediments have been

removed, leaving behind any sediment-dwelling invertebrates. 

The same volume of sediment should be collected and the same number of rinses should be

performed at each sampling location to ensure representativeness between locations. The

number of grabs collected and rinses performed should be recorded on the Benthic

Macroinvertebrate Field Data Sheet (example provided in Attachment 1).

Transfer the sample from the bucket to sample container(s) and preserve in enough 95% ethanol

to cover the sample. Forceps may be needed to remove organisms from the bucket. Place a

sample identification label that includes date, stream name, sampling location, and collector

name into the sample container. The outside of the container should include the same

information and the words "preservative: 95% ethanol". If more that one container is needed for

a sample, each container label should contain all the information for the sample and should be

numbered (e.g., 1 of 2, 2 of 2, etc.). This information will be recorded in the "Sample Log" at

the biological laboratory.

Complete the top portion of the Benthic Macroinvertebrate Field Data Sheet (Attachment 1).

5.2.3 Wadable Waterbodies

Wadable waterbodies (eg: streams, rivers) can be sampled using a kick-net or dip net. Begin

sampling at the downstream end of the reach and proceed upstream. A total of 20 jabs or kicks

will be taken over the length of the reach; a single jab consists of forcefully thrusting the net into

a productive habitat for a linear distance of 0.5m. A kick is a stationary sampling accomplished

by positioning the net and disturbing the substrate for a distance of 0.5m upstream of the net.

Different types of habitat are to be sampled in approximate proportion to their representation of

surface area of the total macroinvertebrate habitat in the reach. For example, if snags comprise

50% of the habitat in a reach and riffles comprise 20%, then 10 jabs should be taken in snag

material and 4 jabs should be take in riffle areas. The remainder of the jabs (6) would be taken

in any remaining habitat type. Habitat types contributing less than 5% of the stable habitat in the

stream reach should not be sampled. In this case, allocate the remaining jabs proportionately

among the predominant substrates. The number of jabs taken in each habitat type should be

recorded on the Benthic Macroinvertebrate Field Data Sheet (example provided in Attachment

1).
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 6

The jabs or kicks collected from the multiple habitats will be composited to obtain a single

homogeneous sample. Every 3 jabs (more often if necessary) wash the collected material by

running clean stream water through the net two to three times, being careful to retain the sample

inside the net. If clogging does occur, discard the material in the net and redo that portion of the

sample in the same habitat type but in a different location. Remove large debris after rinsing and

inspecting it for organisms; place any organisms found into the sample container. Do not spend

time inspecting small debris in the field.

Transfer the sample from the net to sample container(s) and preserve in enough 95% ethanol to

cover the sample. Forceps may be needed to remove organisms from the dip net. Place a sample

identification label that includes date, stream name, sampling location, and collector name into

the sample container. The outside of the container should include the same information and the

words "preservative: 95% ethanol". If more that one container is needed for a sample, each

container label should contain all the information for the sample and should be numbered (e.g., 1

of 2, 2 of 2, etc.). This information will be recorded in the "Sample Log" at the biological

laboratory.

Complete the top portion of the Benthic Macroinvertebrate Field Data Sheet (Attachment 1).

5.3 Sample Collection for Analytical Measurements

Sample collection techniques are identical to those utilized to collect benthic community

samples. The jabs/kicks or dredge samples collected from multiple habitats will be composited

to obtain a single homogeneous sample. Remove any large debris manually and use forceps or

elutriation buckets to extract any organisms from the sample; place any organisms found into the

appropriate sample container as specified in the QAPP. Continue to collect and composite

organisms until the mass requirements for the analytical method are met. Benthic

macroinvertebrate samples that will be analyzed for contaminants should be kept on dry ice.

5.4 Habitat Assessment

Perform habitat assessment after sampling has been completed, and record all observations on

the Habitat Assessment Field Data Sheet (Attachment 1). Having sampled the various

microhabitats and walked the reach helps ensure a more accurate assessment. Conduct the

habitat assessment with another team member, if possible.

Return samples to the laboratory and complete the log-in forms (example provided in

Attachment 2).
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 7

6.0 SAMPLE CONTAINERS AND LABELING

Sample labels must be properly completed, including the sample identification code, date, stream

name, sampling location, and collector's name and placed into the sample container. The outside

of the container should be labeled with the same information. Chain-of-custody forms must

include the same information as the sample container labels. 

7.0 LABORATORY PROCESSING FOR COMMUNITY SAMPLES

7.1 Laboratory Equipment/Supplies

• log-in sheet for samples (example provided in Attachment 2)

• standardized gridded pan (30cm x 36cm) with approximately 30 grids (6cm x 6cm)

• 500 micron sieve

• forceps

• white plastic or enamel pan (15cm x 23cm) for sorting

• specimen vials with caps or stoppers

• sample labels

• benthic macroinvertebrate laboratory bench sheet (example provided in Attachment 2)

• dissecting microscope for organism identification

• fiber optics light source

• compound microscope with phase contrast for identification of mounted organisms (e.g.,

midges)

• 70% ethanol for storage of specimens

• appropriate taxonomic keys

Macroinvertebrate samples should be processed in the laboratory under controlled conditions. 

Aspects of laboratory processing include subsampling, sorting, and identification of organisms.

All samples should be dated and recorded in the "Sample Log" notebook, which is a three-ring

binder book used to store Sample Log forms (Attachment 2) upon receipt by laboratory

personnel. All information from the sample container label must be included on the sample log

sheet. If more than one container was used, the number of containers should be indicated as

well. All samples should be sorted in a single laboratory to enhance quality control.

7.2 Subsampling and Sorting

The Rapid Bioassessment Protocol III uses a fixed-count approach to subsampling and sorting

the organisms from the sample matrix of detritus, sand, and mud. The following protocol is

based on a 200-organism subsample, but it could be used for any subsample size (100, 300, 500,
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 8

etc.). The subsample is sorted and preserved separately from the remaining sample for quality

control checks. 

Prior to processing any samples in a lot (i.e., samples within a collection date, specific

watershed, or project), complete the sample log-in sheet to verify that all samples have arrived at

the laboratory, and are in proper condition for processing.

Thoroughly rinse sample in a 500um mesh sieve to remove preservative and fine sediment. 

Large organic material (whole leaves, twigs, algal or macrophyte mats, etc.) not removed in the

field should be rinsed, visually inspected, and discarded. If the samples have been preserved in

alcohol, it will be necessary to soak the sample contents in water for about 15 minutes to hydrate

the benthic organisms, which will prevent them from floating on the water surface during

sorting. If the sample was stored in more than one container, the contents of all containers for a

given sample should be combined at this time. Gently mix the sample by hand while rinsing to

make homogeneous.

After washing, spread the sample evenly across a pan marked with grids approximately 6cm x

6cm. On the laboratory bench sheet, note the presence of large or obviously abundant

organisms; do not remove them from the pan. 

Use a random numbers table to select 4 numbers corresponding to squares (grids) within the

gridded pan. Remove all material (organisms and debris) from the four grid squares, and place

the material into a shallow white pan and add a small amount of water to facilitate sorting. If

there appear (through a cursory count or observation) to be 200 organisms ± 20% (cumulative of

4 grids), then subsampling is complete. 

Any organism that is lying over a line separating two grids is considered to be on the grid 

containing its head. In those instances where it may not be possible to determine the location of

the head (worms for instance), the organism is considered to be in the grid containing most of its

body.

If the density of organisms is high enough that many more than 200 organisms are contained in

the 4 grids, transfer the contents of the 4 grids to a second gridded pan. Randomly select grids

for this second level of sorting as was done for the first, sorting grids one at a time until 200

organisms ± 20% are found. If picking through the entire next grid is likely to result in a

subsample of greater than 240 organisms, then that grid may be subsampled in the same manner

as before to decrease the likelihood of exceeding 240 organisms. That is, spread the contents of

the last grid into another gridded pan. Pick grids one at a time until the desired number is

reached. The total number of grids for each subsorting level should be noted on the laboratory

bench sheet.
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 9

7.3 Identification of Macroinvertebrates

Taxonomy can be at any level, but should be done consistently among samples. Genus/species

provides more accurate information on ecological/ environmental relationships and sensitivity to

impairment. Family level provides a higher degree of precision among samples and taxonomists,

requires less expertise to perform, and accelerates assessment results. In either case, only those

taxonomic keys that have been peer-reviewed and are available to other taxonomists should be

used. 

Most organisms are identified to the lowest practical level (generally genus or species) by a

qualified taxonomist using a dissecting microscope. Midges (Diptera: Chironomidae) are

mounted on slides in an appropriate medium and identified using a compound microscope. Each

taxon found in a sample is recorded and enumerated in a laboratory bench notebook and then

transcribed to the laboratory bench sheet for subsequent reports. Any difficulties encountered

during identification (e.g., missing gills) are noted on these sheets. 

Labels with specific taxa names (and the taxonomist's initials) are added to the vials of

specimens by the taxonomist. (Note that individual specimens may be extracted from the sample

to be included in a reference collection or to be verified by a second taxonomist.) Slides are

initialed by the identifying taxonomist. A separate label may be added to slides to include the

taxon (taxa) name(s) for use in a voucher or reference collection.

Record the identity and number of organisms on the Laboratory Bench Sheet (Attachment 2). 

Either a tally counter or "slash" marks on the bench sheet can be used to keep track of the

cumulative count. Also, record the life stage of the organisms, the taxonomist's initials and the

Taxonomic Certainty Rating (TCR) as a measure of confidence.

In the spaces provided on the bench sheet, explain certain TCR ratings or condition of

organisms. Other comments can be included to provide additional insights for data

interpretation. If QC was performed, record on the back of the bench sheet.

For archiving samples, specimen vials, (grouped by station and date), are placed in jars with a

small amount of denatured 70% ethanol and tightly capped. The ethanol level in these jars must

be examined periodically and replenished as needed, before ethanol loss from the specimen vials

takes place. A stick-on label is placed on the outside of the jar indicating sample identifier, date,

and preservative (denatured 70% ethanol).

All samples should be stored on wet ice (4o

C) in a secured cooler. Ship samples under chainof-custody, protected with suitable resilient packing material to reduce shock, vibration, and

disturbance. 
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 10

8.0 SUBSAMPLE PROCEDURE MODIFICATIONS

As an improvement to the mechanics of the technique, a sorting tray was designed that consists

of two parts, a rectangular plastic or plexiglass pan (36cm x 30cm) with a rectangular sieve

insert. The sample is placed on the sieve, in the pan and dispersed evenly. 

When a random grid(s) is selected, the sieve is lifted to temporarily drain the water. A

"cookie-cutter" like metal frame 6cm x 6cm is used to clearly define the selected grid; debris

overhanging the grid may be cut with scissors. A 6cm flat scoop is used to remove all debris and

organisms from the grid. The contents are then transferred to a separate sorting pan with water

for removal of macroinvertebrates.

These modifications have allowed for rapid isolation of organisms within the selected grids and

easy removal of all organisms and debris within a grid while eliminating investigator bias. Save

the sorted debris residue in a separate container. Add a label that includes the words "sorted

residue" in addition to all prior sample label information and preserve in 95% ethanol. Save the

remaining unsorted sample debris residue in a separate container labeled "sample residue"; this

container should include the original sample label. Length of storage and archival is determined

by the laboratory or benthic section supervisor.

Place the sorted 200-organism (± 20%) subsample into glass vials, and preserve in 70% ethanol. 

Label the vials inside with the sample identifier or lot number, date, stream name, sampling

location and taxonomic group. If more than one vial is needed, each should be labeled separately

and numbered (e.g., 1 of 2, 2 of 2). For convenience in reading the labels inside the vials, insert

the labels left-edge first. If identification is to occur immediately after sorting, a petri dish or

watch glass can be used instead of vials.

Midge (Chironomidae) larvae and pupae should be mounted on slides in an appropriate medium

(e.g., Euperal, CMC-9); slides should be labeled with the site identifier, date collected, and the

first initial and last name of the collector. As with midges, worms (Oligochaeta) must also be

mounted on slides and should be appropriately labeled.

Fill out header information on Laboratory Bench Sheet (see Attachment 2). Also check

subsample target number. Complete back of sheet for subsampling/sorting information. Note

number of grids picked, time expenditure, and number of organisms. If QC check was

performed on a particular sample, person conducting QC should note findings on the back of the

Laboratory Bench Sheet. Calculate sorting efficiency to determine whether sorting effort passes

or fails.
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 11

Record date of sorting and slide monitoring, if applicable, on Log-In Sheet as documentation of

progress and status of completion of sample lot.

9.0 DECONTAMINATION

After sampling has been completed at a given site, all nets, pans, etc. that have come in contact

with the sample should be rinsed thoroughly, examined carefully, and picked free of organisms

or debris. Any additional organisms found should be placed into the sample containers. The

equipment should be examined again prior to use at the next sampling site.

Excess sediment and substrate material not included in the sample should be washed into the

stream, pond, lake, or surface impoundment where it came from. All marker flags (if reused)

should be decontaminated by wiping off with towels and/or baby wipes before re-use. 

Throw all used wipes and gloves into the trash bags and take with you to dispose of at the field

office.

10.0 RECORD KEEPING AND QUALITY CONTROL

Each field crew will carry a three-ring binder book that contains the Benthic Macroinvertebrate

Field Data Sheet, Physical Characterization/Water Quality Field Data Sheet, and sample labels. 

In addition, a field notebook should be maintained by each individual or team that is collecting

samples, as described in the Project Plan. Each sampling location must be recorded on the site

diagram. Each sample should have an ID number affixed to the outside of the wide-mouth

bottle, and the duplicate label must be affixed to the sample data sheet. Deviations from this

sampling plan should be noted in the field notebook, as necessary.

10.1 Required Information

For each location, the notebook information must include:

a. date

b. time

c. personnel

d. weather conditions

e. sample identification numbers that were used

f. descriptions of any deviations to the Project Plan and the reason for the deviation

Samples taken from waters with visible color abnormalities, foaming, unusual odor, iridescent

film, or other indications of non-homogeneous conditions should also be noted. Field personnel
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003 Page 12

will collect the proper type and quantity of quality control samples as prescribed in the QAPP. 

10.2 Field Quality Control Samples

The type of quality control samples, and the frequency of collection, are specified in the QAPP. 

The following quality control samples will be collected.

Field Duplicate: Field duplicate samples are collected at the same time as the primary

sample, and are used to evaluate precision and reproducibility of the analysis and

sampling technique or collection team. In this case, the field duplicate sample is a

second sample of benthic macroinvertebrates collected from the same reach or widths of

a stream.

11.0 REFERENCES

Barbour, M.T., J. Gerritsen, B.D. Snyder, and J.B. Stribling. 1999. Rapid Bioassessment

Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic Macroinvertebrates and

Fish, Second Edition. EPA 841-B-99-002. U.S. Environmental Protection Agency; Office of

Water; Washington, D.C.

USEPA. 2003. Sampling and Analytical Procedures for GLNPO’s Open Lake Water Quality

Survey of the Great Lakes; Chapter 4 - Biological Parameters; LG406 - Standard Operating

Procedure for Benthic Invertebrate Field Sampling Procedure, Revision 07. EPA 905-R-03-002. 

March 2003.
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

ATTACHMENT 1
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

Benthic Macroinvertebrate Field Data Sheet 
BENTHIC MACROINVERTEBRATE FIELD DATA SHEET

STREAM NAME LOCATION

STATION #_________ RIVERMILE_________ STREAM CLASS

LAT ______________ LONG ______________ RIVER BASIN

STORET # AGENCY

INVESTIGATORS LOT NUMBER

FORM COMPLETED BY DATE _______

TIME_______ AM PM

REASON FOR SURVEY

HABITAT

TYPES

Indicate the percentage of each habitat type present

‘ Cobble_____% ‘ Snags_____% ‘ Vegetated Banks_____% ‘ Sand_____%

‘ Submerged Macrophytes_____% ‘ Other ( )_____%

SAMPLE

COLLECTION

Gear used ‘ D-frame ‘ kick-net ‘ Other _________________________

How were the samples collected? ‘ wading ‘ from bank ‘ from boat

Indicate the number of jabs/kicks taken in each habitat type.

‘ Cobble_____ ‘ Snags_____ ‘ Vegetated Banks_____ ‘ Sand_____

‘ Submerged Macrophytes_____ ‘ Other ( )_____

GENERAL

COMMENTS

QUALITATIVE LISTING OF AQUATIC BIOTA

Indicate estimated abundance: 0 = Absent/Not Observed, 1 = Rare, 2 = Common, 3= Abundant, 4 = Dominant

Periphyton 0 1 2 3 4 Slimes 0 1 2 3 4

Filamentous Algae 0 1 2 3 4 Macroinvertebrates 0 1 2 3 4

Macrophytes 0 1 2 3 4 Fish 0 1 2 3 4

FIELD OBSERVATIONS OF MACROBENTHOS

Indicate estimated abundance: 0 = Absent/Not Observed, 1 = Rare (1-3 organisms), 2 = Common (3-9

organisms), 3= Abundant (>10 organisms), 4 = Dominant (>50 organisms)

Porifera 0 1 2 3 4 Anisoptera 0 1 2 3 4 Chironomidae 01234

Hydrozoa 0 1 2 3 4 Zygoptera 0 1 2 3 4 Ephemeroptera 0 1 2 3 4

Platyhelminthes 0 1 2 3 4 Hemiptera 0 1 2 3 4 Trichoptera 01234

Turbellaria 0 1 2 3 4 Coleoptera 0 1 2 3 4 Other 0 1 2 3 4

Hirudinea 0 1 2 3 4 Lepidoptera 01234

Oligochaeta 0 1 2 3 4 Sialidae 0 1 2 3 4

Isopoda 0 1 2 3 4 Corydalidae 0 1 2 3 4

Amphipoda 0 1 2 3 4 Tipulidae 0 1 2 3 4

Decapoda 0 1 2 3 4 Empididae 0 1 2 3 4

Gastropoda 0 1 2 3 4 Simuliidae 01234

Bivalvia 0 1 2 3 4 Tabinidae 0 1 2 3 4

Culcidae 0 1 2 3 4
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

Physical Characterization/Water Quality Field Data Sheet
PHYSICAL CHARACTERIZATION/WATER QUALITY FIELD DATA SHEET

(Pg. 1)

STREAM NAME LOCATION

STATION #_________ RIVERMILE_________ STREAM CLASS

LAT ______________ LONG ______________ RIVER BASIN

STORET # AGENCY

INVESTIGATORS

FORM COMPLETED BY DATE ________

TIME _________ AM PM

REASON FOR SURVEY

WEATHER

CONDITIONS

Now

‘

‘

‘

____%‘

‘

storm (heavy rain)

rain (steady rain)

showers (intermittent)

%cloud cover

clear/sunny

Past 24

hours

‘

‘

‘

____%

‘

‘

Has there been a heavy rain in the last 7 days?

‘ Yes ‘ No

Air Temperature_____0

 C

Other_________________________________

SITE LOCATION AND

MAP

Draw a map of the site and indicate the areas sampled (or attach a photograph)

STREAM

CHARACTERIZATION Stream Subsystem

‘ Perennial ‘ Intermittent ‘ Tidal

Stream Origin

‘ Glacial ‘ Spring-fed ‘ Non-glacial montane ‘ Mixture of origins ‘ Swamp and bog ‘ Other__________ 

Stream Type

‘ Coldwater ‘ Warmwater

Catchment Area__________km2
PHYSICAL CHARACTERIZATION/WATER QUALITY FIELD DATA SHEET

(Pg. 2)

WATERSHED

FEATURES

Predominant Surrounding Landuse

‘ Forest ‘ Commercial

‘ Field/Pasture ‘ Industrial

‘ Agricultural ‘ Other _______________ ‘ Residential

Local Watershed NPS Pollution

‘ No evidence ‘ Some potential sources ‘ Obvious sources

Local Watershed Erosion

‘ None ‘ Moderate ‘ Heavy

RIPARIAN

VEGETATION

(18 meter buffer)

Indicate the dominant type and record the dominant species present ‘ Trees ‘ Shrubs ‘ Grasses ‘ Herbaceous

dominant species present __________________________________________________

INSTREAM 

FEATURES

Estimated Reach Length _______m

Estimated Stream Width _______m

Sampling Reach Area _______m2

Area in km2 (m2x1000) _______km2

Estimated Stream Depth _______m

Surface Velocity _______m/sec

(at thalweg)

Canopy Cover

‘ Partly open ‘ Partly shaded ‘ Shaded

High Water Mark _______m

Proportion of Reach Represented by Stream

Morphology Types

‘ Riffle_______% ‘ Run_______% ‘ Pool_______%

Channelized ‘ Yes ‘ No

Dam Present ‘ Yes ‘ No

LARGE WOODY 

DEBRIS

LWD _______m2

Density of LWD _______m2/km2 (LWD/ reach area)

AQUATIC

VEGETATION

Indicate the dominant type and record the dominant species present ‘ Rooted emergent ‘ Rooted submergent ‘ Rooted floating ‘ Free floating ‘ Floating Algae ‘ Attached Algae

dominant species present __________________________________________________

Portion of the reach with aquatic vegetation _____%

WATER QUALITY Temperature________0

 C

Specific Conductance________

Dissolved Oxygen ________

pH ________

Turbidity ________

WQ Instrument Used _______________

Water Odors

‘ Normal/None ‘ Sewage ‘ Petroleum ‘ Chemical

‘ Fishy ‘ Other________________

Water Surface Oils

‘ Slick ‘ Sheen ‘ Globs ‘ Flecks

‘ None ‘ Other_________________________

Turbidity (if not measured)

‘ Clear ‘ Slightly turbid ‘ Turbid

‘ Opaque ‘ Stained

‘ Other________

SEDIMENT/

SUBSTRATE

Odors

‘ Normal ‘ Sewage ‘ Petroleum

‘ Chemical ‘ Anaerobic ‘ None

‘Other______________________________

Oils

‘ Absent ‘ Slight

‘ Moderate ‘ Profuse

Deposits

‘ Sludge ‘ Sawdust ‘ Paper fiber ‘ Sand

‘ Relict shells ‘ Other_______________

Looking at stones which are not deeply embedded,

are the undersides black in color?

‘ Yes ‘ No
PHYSICAL CHARACTERIZATION/WATER QUALITY FIELD DATA SHEET

(Pg. 3)

INORGANIC SUBSTRATE COMPONENTS

(should add up to 100%)

ORGANIC SUBSTRATE COMPONENTS

(does not necessarily add up to 100%)

Substrate

Type

Diameter % Composition in

Sampling Reach

Substrate

Type

Characteristic % Composition in

Sampling Area

Bedrock Detritus sticks, wood, coarse plant

materials (CPOM) Boulder > 256 mm (10")

Cobble 64-256 mm (2.5"-10") Muck-Mud black, very fine organic

(FPOM) Gravel 2-64 mm (0.1"-2.5")

Sand 0.06-2mm (gritty) Marl grey, shell fragments

Silt 0.004-0.06 mm

Clay < 0.004 mm (slick)
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

Habitat Assessment Field Data Sheet 
HABITAT ASSESSMENT FIELD DATA SHEET - LOW GRADIENT STREAMS

STREAM NAME LOCATION

STATION #_______ RIVERMILE__________ STREAM CLASS

LAT _______________ LONG _______________ RIVER BASIN

STORET # AGENCY

INVESTIGATORS

FORM COMPLETED BY DATE _________

TIME _________ AM PM

REASON FOR SURVEY

Parameters to be evaluated in sampling reach

Habitat

Parameter

Condition Category

Optimal Suboptimal Marginal Poor

1. Epifaunal

Substrate/

Available Cover

Greater than 50% of

substrate favorable for

epifaunal colonization

and fish cover; mix of

snags, submerged logs,

undercut banks, cobble or

other stable habitat and at

stage to allow full

colonization potential

(i.e., logs/snags that are

not new fall and not

transient).

30-50% mix of stable

habitat; well-suited for

full colonization

potential; adequate

habitat for maintenance

of populations; presence

of additional substrate in

the form of newfall, but

not yet prepared for

colonization (may rate at

high end of scale).

10-30% mix of stable

habitat; habitat

availability less than

desirable; substrate

frequently disturbed or

removed.

Less than 10% stable

habitat; lack of habitat is

obvious; substrate

unstable or lacking.

SCORE 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

2. Pool Substrate

Characterization

Mixture of substrate

materials, with gravel and

firm sand prevalent; root

mats and submerged

vegetation common.

Mixture of soft sand,

mud, or clay; mud may

be dominant; some root

mats and submerged

vegetation present.

All mud or clay or sand

bottom; little or no root

mat; no submerged

vegetation.

Hard-pan clay or

bedrock; no root mat or

vegetation.

SCORE 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

3. Pool Variability

Even mix of largeshallow, large-deep,

small-shallow, smalldeep pools present.

Majority of pools largedeep; very few shallow.Shallow pools much

more prevalent than deep

pools.

Majority of pools smallshallow or pools absent.

SCORE 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

4. Sediment

Deposition

Little or no enlargement

of islands or point bars

and less than <20% of

the bottom affected by

sediment deposition. 

Some new increase in bar

formation, mostly from

gravel, sand or fine

sediment; 20-50% of the

bottom affected; slight

deposition in pools. 

Moderate deposition of

new gravel, sand or fine

sediment on old and new

bars; 50-80% of the

bottom affected;

sediment deposits at

obstructions, 

constrictions, and bends;

moderate deposition of

pools prevalent.

Heavy deposits of fine

material, increased bar

development; more than

80% of the bottom

changing frequently;

pools almost absent due

to substantial sediment

deposition.

SCORE 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

5. Channel Flow

Status

Water reaches base of

both lower banks, and

minimal amount of

channel substrate is

exposed.

Water fills >75% of the

available channel; or

<25% of channel

substrate is exposed.

Water fills 25-75% of the

available channel, and/or

riffle substrates are

mostly exposed.

Very little water in

channel and mostly

present as standing pools.

SCORE 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
Parameters to be evaluated broader than sampling reach

Habitat

Parameter

Condition Category

Optimal Suboptimal Marginal Poor

6. Channel

Alteration 

Channelization or

dredging absent or

minimal; stream with

normal pattern.

Some channelization

present, usually in areas

of bridge abutments;

evidence of past

channelization, i.e.,

dredging, (greater than

past 20 yr) may be

present, but recent

channelization is not

present.

Channelization may be

extensive; embankments

or shoring structures

present on both banks;

and 40 to 80% of stream

reach channelized and

disrupted.

Banks shored with

gabion or cement; over

80% of the stream reach

channelized and

disrupted. Instream

habitat greatly altered or

removed entirely.

SCORE 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

7. Channel

Sinuosity

The bends in the stream

increase the stream

length 3 to 4 times longer

than if it was in a straight

line. (Note - channel

braiding is considered

normal in coastal plains

and other low-lying

areas. This parameter is

not easily rated in these

areas.)

The bends in the stream

increase the stream

length 1 to 2 times longer

than if it was in a straight

line.

The bends in the stream

increase the stream

length 1 to 2 times longer

than if it was in a straight

line.

Channel straight;

waterway has been

channelized for a long

distance.

SCORE 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

8. Bank Stability

(score each bank)

Banks stable; evidence of

erosion or bank failure

absent or minimal; little

potential for future

problems. <5% of bank

affected.

Moderately stable;

infrequent, small areas of

erosion mostly healed

over. 5-30% of bank in

reach has areas of

erosion.

Moderately unstable; 30-

60% of bank in reach has

areas of erosion; high

erosion potential during

floods.

Unstable; many eroded

areas; "raw" areas

frequent along straight

sections and bends;

obvious bank sloughing;

60-100% of bank has

erosional scars.

SCORE ___ (LB) Left Bank 10 9 8 7 6 5 4 3 2 1 0

SCORE ___ (RB) Right Bank 10 9 8 7 6 5 4 3 2 1 0

9. Vegetative

Protection (score

each bank)

Note: determine left

or right side by

facing downstream.

More than 90% of the

streambank surfaces and

immediate riparian zone 

covered by native

vegetation, including

trees, understory shrubs,

or nonwoody

macrophytes; vegetative

disruption through

grazing or mowing

minimal or not evident;

almost all plants allowed

to grow naturally.

70-90% of the

streambank surfaces

covered by native

vegetation, but one class

of plants is not wellrepresented; disruption

evident but not affecting

full plant growth

potential to any great

extent; more than onehalf of the potential plant

stubble height remaining.

50-70% of the

streambank surfaces

covered by vegetation;

disruption obvious;

patches of bare soil or

closely cropped

vegetation common; less

than one-half of the

potential plant stubble

height remaining.

Less than 50% of the

streambank surfaces

covered by vegetation;

disruption of streambank

vegetation is very high;

vegetation has been

removed to 

5 centimeters or less in

average stubble height.

SCORE ___ (LB) Left Bank 10 9 8 7 6 5 4 3 2 1 0

SCORE ___ (RB) Right Bank 10 9 8 7 6 5 4 3 2 1 0

10. Riparian

Vegetative Zone

Width (score each

bank riparian zone)

Width of riparian zone

>18 meters; human

activities (i.e., parking

lots, roadbeds, clear-cuts,

lawns, or crops) have not

impacted zone.

Width of riparian zone

12-18 meters; human

activities have impacted

zone only minimally.

Width of riparian zone 6-

12 meters; human

activities have impacted

zone a great deal.

Width of riparian zone

<6 meters: little or no

riparian vegetation due to

human activities.

SCORE ___ (LB) Left Bank 10 9 8 7 6 5 4 3 2 1 0

SCORE ___ (RB) Right Bank 10 9 8 7 6 5 4 3 2 1 0

Total Score __________
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

ATTACHMENT 2
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

Sample Log-in Sheet
page __ of __

BENTHIC MACROINVERTEBRATE SAMPLE LOG-IN SHEET

Date

Collected

Collected

By

Number of

Containers

Preservation Station

#

Stream Name and Location Date

Received by

Lab

Lot Number Date of Completion

sorting mounting identification
TECHNICAL STANDARD OPERATING PROCEDURE

BENTHIC MACROINVERTEBRATE SAMPLING & PROCESSING

SOP #EH-04, East Helena Site, Montana

September 2003

Benthic Macroinvertebrate Laboratory Bench Sheet
BENTHIC MACROINVERTEBRATE LABORATORY BENCH SHEET (Pg. 1)

page _____ of _____

STREAM NAME LOCATION

STATION #_________ RIVERMILE_________ STREAM CLASS 

LAT ______________ LONG ______________ RIVER BASIN

STORET # AGENCY

COLLECTED BY DATE_________ LOT #

TAXONOMIST DATE_________ SUBSAMPLE TARGET ‘ 100 ‘ 200 ‘ 300 ‘ Other ____ 

Enter Family and/or Genus and Species name on blank line. 

Organisms No. LS TI TCR Organisms No. LS TI TCR

Oligochaeta Megaloptera

Hirudinea Coleoptera

Isopoda

Amphipoda Diptera

Decapoda

Ephemeroptera

Gastropoda

Pelecypoda

Plecoptera

Other

Trichoptera

Hemiptera

Taxonomic certainty rating (TCR) 1-5:1=most certain, 5=least certain. If rating is 3-5, give reason (e.g., missing gills). 

LS= life stage: I = immature; P = pupa; A = adult TI = Taxonomists initials

Total No. Organisms _______________ Total No. Taxa _______________
( ) # organisms 

recovered by 

checker

# organisms 

originally sorted

% sorting 

efficiency

# organisms 

originally sorted

+ .

. =

BENTHIC MACROINVERTEBRATE LABORATORY BENCH SHEET (Pg. 2)

SUBSAMPLING/SORTING

INFORMATION

Sorter ___________________

Date ___________________

Number of grids picked: __________

Time expenditure __________ No. of organisms __________

Indicate the presence of large or obviously abundant organisms:

QC: ‘ YES ‘ NO QC Checker

_________________________

$90%, sample passes __________

<90%, sample fails, action taken ____________________________________

________________________________________________________________

TAXONOMY

ID ___________________

Date ___________________

Explain TCR ratings of 3-5:

Other Comments (e.g. condition of specimens):

QC: ‘ YES ‘ NO QC Checker

_________________________

Organism recognition ‘ pass ‘ fail

Verification complete ‘ YES ‘ NO