BENTHIC MACROINVERTEBRATES (10500)/Introduction 10-63

10500 BENTHIC MACROINVERTEBRATES*

10500 A. Introduction

1. Definition 2. Response to Environment

Benthic macroinvertebrates are animals inhabiting the sedi- The species composition and population or species density
ment, or living on or in other available bottom substrates of (numbers of individuals per unit area) of macroinvertebrate
freshwater, estuarine, and marine ecosystems. During all or part communities in streams, lakes, estuaries, and marine waters can
of their life cycles, these organisms may construct attached be uniform from year to year in unperturbed environments.
cases, tubes, or nets that they live on or in; roam freely over However, life-cycle dynamics produce variations in species
rocks, organic debris, and other substrates; or burrow freely in composition and abundance either temporally or spatially.
substrates. Although they vary in size from small forms, difficult Most aquatic habitats, particularly free-flowing streams and
to see without magnification, to other individuals large enough to waters with acceptable water quality and substrate conditions,
see without difficulty, macroinvertebrates are considered histor- support diverse macroinvertebrate communities in which there is
ically by definition to be visible to the unaided eye and retained a reasonably balanced distribution of species among the total
on a U.S. Standard No. 30 sieve (0.595-mm or 0.600-mm open- number of individuals present. Such communities respond to
ings).1 changing habitats and water quality by alterations in community
The standard sieve for collecting freshwater, estuarine, and structure (invertebrate abundance and composition). However,
marine benthic macroinvertebrates is the U.S. Standard No. 30 many habitats, especially disturbed ones, may be dominated by
sieve; however, some estuarine and marine programs use the a few species.
U.S. Standard No. 50 sieve (0.300-mm openings) or the U.S. Macroinvertebrate community responses to environmental
Standard No. 35 sieve (0.500-mm openings). For all aquatic changes are useful in assessing the impact of municipal, indus-
assessment programs, use of the No. 30 sieve to collect benthic trial, oil, and agricultural wastes, and impacts from other land
fauna of freshwater, estuarine, and marine habitats or from any uses on surface waters. Four types of environmental changes for
water transport system is recommended. To accommodate some which patterns of macroinvertebrate community structure
old historical databases and if the data-quality objectives of the change have been documented are: increased inorganic nutrients,
study permit, a U.S. Standard No. 28 sieve (1.0-mm openings) increased organic loading, substrate alteration, and toxic chem-
might be utilized. To obtain a more representative sample of the ical pollution. Inorganic nutrients and severe organic pollution
benthos that would include smaller forms or early life-stages, usually result in a reduction in the variety of macroinvertebrates
and other taxa of macroinvertebrates, a U.S. Standard No. 60 to only the most tolerant ones and a corresponding increase in
sieve (0.250-mm openings) may be used. density of those tolerating the polluted conditions, usually asso-
The standardization of bioassessment for species composition, ciated with low dissolved oxygen concentration. In some cases
taxa richness, diversity, evenness, trophic levels, and major severe organic pollution, siltation, or toxic chemical pollution
taxonomic spatial and temporal patterns may be enhanced sig- may reduce or even eliminate the entire macroinvertebrate com-
nificantly by the conventional use of a U.S. Standard No. 30 munity from an affected area. Not all cases conform to those
sieve. described because conditions may be mediated by other envi-
The major macroinvertebrates found in freshwater are flat- ronmental (biological, chemical, and physical) conditions.
worms, annelids, mollusks, crustaceans, and insects. The major Assessing the impact of a pollution source generally involves
macroinvertebrate groups included in estuarine and marine wa- comparing macroinvertebrate communities and their physical
habitats at sites influenced by pollution with those collected from
ters are bryozoans, sponges, annelids, mollusks, roundworms,
adjacent unaffected sites. This can include a gradient away from
cnidarians (coelenterates), crustaceans, insects, and echino-
point sources of contamination. The procedure includes sam-
derms.
pling and analyzing types of communities from different sites
and subsequently determining whether the presumed pollution-
affected community differs from the presumed nonaffected com-
* Approved by Standard Methods Committee, 2001.
Joint Task Group: Donald J. Klemm (chair), David C. Beckett, Peter M. Chapman, munity. The basic information required for most community
Philip A. Lewis, Morris H. Roberts, Jr., Don W. Schloesser, William T. Thoeny. structure analyses is a count of individuals per species. From the
10-64 BIOLOGICAL EXAMINATION (10000)

count data, communities can be characterized and compared 4. Bibliography

according to community structure, density, diversity, community
metrics, pollution indicators, or other analyses,1 including vari- HYNES, H.B.N. 1963. The Biology of Polluted Waters. Liverpool Univ.
ous statistical methods (see 10500D). Biomass and productivity Press, England.
estimates also can be determined with the organisms collect- MACAN, T.T. 1963. Freshwater Ecology. John Wiley & Sons, New York,
ed.1– 4 Equally desirable is a characterization of the dissolved N.Y.
oxygen concentration, substrate, water depth, type of sediment, RUTTNER, F. 1966. Fundamentals of Limnology. Univ. Toronto Press,
grain size of the sediment, total organic carbon (TOC), and other Toronto, Ont.
site- and situation-specific characteristics. MACKENTHUN, K.M. & W.M. INGRAM. 1967. Biological Associated Prob-
While the following macroinvertebrate methods traditionally lems in Freshwater Environments. Federal Water Pollution Control
Admin., Washington, D.C.
are used for sampling and quantifying benthic invertebrate com-
HYNES, H.B.N. 1970. The Ecology of Running Waters. Univ. Toronto
munities, other methods also are being evaluated in an effort to
Press, Toronto, Ont.
develop and implement narrative biological criteria for surface
ODUM, E.P. 1971. Fundamentals of Ecology, 3rd ed. Saunders Publish-
waters.5 Not discussed here are EPA-developed rapid bioassess- ing Co., Philadelphia, Pa.
ment techniques6,7 and Environmental Monitoring and Assess- COULL, B.C., ed. 1977. Ecology of Marine Benthos. University of South
ment Program (EMAP) protocols for field operations and meth- Carolina Press, Columbia.
ods and laboratory methods for sampling macroinvertebrates and PEARSON, T.H. & R. ROSENBERG. 1978. Macrobenthic succession in
assessing the ecological conditions of wadeable streams.8 –10 relation to organic enrichment and pollution of the marine environ-
ment. Oceanogr. Mar. Biol. Annu. Rev. 16:229.
RESH, V.H. & D.M. ROSENBERG. 1984. The Ecology of Aquatic Insects.
3. References Praeger Publ., New York, N.Y.
BROWER, J.R. & J.H. ZAR. 1984. Field and Laboratory Methods for
1. KLEMM, D.J., P.A. LEWIS, F. FULK & J.M. LAZORCHAK. 1990. Mac- General Ecology. Wm. C. Brown Publ., Dubuque, Iowa.
roinvertebrate Field and Laboratory Methods for Evaluating the WARD, J.V. 1984. Ecological perspectives in the management of aquatic
Biological Integrity of Surface Waters. EPA-600/4-90-030. Envi- insects. In V.H. Resh & D.M. Rosenberg, eds. The Ecology of
ronmental Monitoring Systems Laboratory, U.S. Environmental Aquatic Insects. p. 558. Praeger Scientific, New York, N.Y.
Protection Agency, Cincinnati, Ohio.
WIEDERHOLM, T. 1984. Responses of aquatic insects to environmental
2. WETZEL, R.G. & G.E. LIKENS. 1991. Limnological Analyses, 2nd ed.
pollution. In V.H. Resh & D.M. Rosenberg, eds. The Ecology of
Springer-Verlag, New York, N.Y.
Aquatic Insects, p. 508. Praeger Scientific, New York, N.Y.
3. PLANTE, C. & J.A. DOWNING. 1989. Production of freshwater inver-
CUMMINS, K.W. & M.A. WILZBACH. 1985. Field Procedures for Analysis
tebrate populations in lakes. Can. J. Fish. Aquat. Sci. 46:1489.
4. PLANTE, C. & J.A. DOWNING. 1990. Empirical evidence for differ- of Functional Feeding Groups of Stream Macroinvertebrates. Con-
ences among methods for calculating secondary production. J. N. tribution 1611. Appalachian Environ. Lab., Univ. Maryland, Frostburg.
Amer. Benthol. Soc. 9(1):9. LEONARD, P.M. & D.J. ORTH. 1986. Application and testing of an index
5. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1996. Biological Criteria. of biotic integrity in small, coolwater streams. Trans. Amer. Fish.
Technical Guidance for Streams and Small Rivers. G.R. Gibson, Jr., Soc. 115:401.
ed. EPA-822-B-96-001, Off. Water, U.S. Environmental Protection WARWICK, R.M. 1986. A new method for determining pollution effects
Agency, Washington, D.C. on marine macrobenthic communities. Mar. Biol. 92:557.
6. PLAFKIN, J.L., M.T. BARBOUR, K.D. PORTER, S.K. GROSS & R.M. HILSENHOFF, W.L. 1987. An improved biotic index of organic stream
HUGHES. 1989. Rapid Bioassessment Protocols for Use in Streams pollution. Great Lakes Entomol. 20:31.
and Rivers. Benthic Macroinvertebrates and Fish. EPA-440/4-89- ROHM, C.M., J.W. GIESE & C.C. BENNETT. 1987. Evaluation of aquatic
001, Off. Water, U.S. Environmental Protection Agency, Washing- ecoregion classification of streams in Arkansas. Freshwater Ecol.
ton, D.C. 4:127.
7. BARBOUR, M.T., J. GERRITSEN, B.D. SNYDER & J.B. STRIBLING. 1999. SHACKELFORD, B. 1988. Rapid Bioassessments of Lotic Macroinverte-
Rapid Bioassessment Protocols for Use in Streams and Wadeable brate Communities: Biocriteria Development. Arkansas Dep. Pol-
Rivers: Periphyton, Benthic Macroinvertebrates, and Fish. EPA lution Control & Ecology, Little Rock.
841-B-99-002, Off. Water, U.S. Environmental Protection Agency, STEEDMAN, R.J. 1988. Modification and assessment of an index of biotic
Washington, D.C. integrity to quantify stream quality in southern Ontario. Can. J.
8. LAZORCHAK, J.M., D.J. KLEMM & D.V. PECK. 1998. Environmental Fish. Aquat. Sci. 45:492.
Monitoring and Assessment Program-Surface Waters: Field Oper- WHITTIER, T.R., R.M. HUGHES & D.P. LARSEN. 1988. Correspondence
ations and Methods for Measuring the Ecological Conditions of
between ecoregions and spatial patterns in stream ecosystems in
Wadeable Streams. EPA 620-R-94-004F, National Exposure Re-
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search Lab., U.S. Environmental Protection Agency, Cincinnati,
U.S. ENVIRONMENTAL PROTECTION AGENCY. 1988. Proc. 1st National Work-
Ohio and National Health & Environmental Effects Research Lab.,
U.S. Environmental Protection Agency, Corvallis, Ore. shop on Biological Criteria, Lincolnwood, Ill., Dec. 2-4, 1987. Rep. No.
9. KLEMM, D.J. & J.M. LAZORCHAK, eds. 1994 (draft). Environmental 905/9-89/003, U.S. Environmental Protection Agency, Chicago, Ill.
Monitoring and Assessment Program Surface Waters and Region 3 OHIO ENVIRONMENTAL PROTECTION AGENCY. 1989. Biological Criteria for
Regional Environmental Monitoring and Assessment Program, the Protection of Aquatic Life: Volume III. Standardized Biological
1994 Pilot Laboratory Methods Manual for Streams. EPA 620-R- Field Sampling and Laboratory Methods for Assessing Fish and
94-003. National Exposure Research Lab., U.S. Environmental Pro- Macroinvertebrate Communities. Ohio Environmental Protection
tection Agency, Cincinnati, Ohio. Agency, Columbus.
10. BARBOUR, M.T. 1997. Perspectives from North America on the REYNOLDSON, T.B., D.W. SCHLOESSER & B.A. MANNY. 1989. Develop-
programmatic and scientific elements of an effective bioassessment. ment of a benthic invertebrate objective for mesotrophic Great
Human Ecol. Risk Assess. 3:929. Lakes waters. J. Great Lakes Res. 15:669.
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BURD, B.J., A. NEMEC & R.O. BRINKHURST. 1990. The development and 001, Off. Science & Technology, U.S. Environmental Protection
application of analytical methods in benthic marine infaunal stud- Agency, Washington, D.C.
ies. Advan. Mar. Biol. 26:169. PEPPER, I.L., C.P. GERBA & M.L. BRUSSEAU. 1996. Pollution Science.
NATIONAL RESEARCH COUNCIL. 1990. Managing Troubled Waters: The Academic Press, San Diego, Calif.
Role of Marine Environmental Monitoring. National Academy PATRICK, R. 1996. Rivers of the United States: Vol. III: The Eastern and
Press, Washington, D.C. Southeastern States. John Wiley & Sons, New York, N.Y.
WESTON, D. 1990. Quantitative examination of macrobenthic changes BARBOUR, M.T. 1997. The re-invention of biological assessment in the
along an organic enrichment gradient. Mar. Ecol. Prog. Ser. 61:233. U.S. Human Ecol. Risk Assess. 3:933.
YOUNT, J.D. & G.J. NIEMI. 1990. Recovery of lotic communities and U.S. ENVIRONMENTAL PROTECTION AGENCY. 1997. Priorities for Ecological
ecosystems from disturbance—a narrative review of case studies.
Protection: An Initial List and Discussion Document for EPA. EPA
Environ. Manag. 14:547.
600-S-97-002, Off. Research & Development, Washington, D.C.
KARR, J.R. & B.L. KERANS. 1992. Components of biological integrity:
U.S. ENVIRONMENTAL PROTECTION AGENCY. 1998. Lake and Reservoir
their definition and use in development of an invertebrate IBI. In
T.P. Simon & W.S. David, eds. Environmental Indicators: Mea- Bioassessment and Biocriteria. Technical Guidance Document,
surement and Assessment Endpoints, Chapter 1. EPA 905-R-92- EPA 841-B-98-007, Off. Water, U.S. Environmental Protection
003, U.S. Environmental Protection Agency, Chicago, Ill. Agency, Washington, D.C.
BLAKE, J.A. & A. LISSNER. 1993. Taxonomic atlas of the benthic fauna PATRICK, R. 1998. The Mississippi River and Its Tributaries North of St.
of the Santa Maria Basin and Western Santa Barbara Channel, Vol. Louis. Vol. IV, Part A. John Wiley & Sons, New York, N.Y.
1. Santa Barbara Mus. Natural History, Santa Barbara, Calif. PATRICK, R. 1998. The Mississippi River and Its Tributaries South of St.
ROSENBERG, D.M. & V.H. RESH, eds. 1993. Freshwater Biomonitoring Louis. Vol. IV, Part B. John Wiley & Sons, New York, N.Y.
and Benthic Macroinvertebrates. Chapman-Hall, New York, N.Y. MACBROOM, J.G. 1998. The River Book. Connecticut Dep. Environmen-
CAIRNS, J., JR. 1993. A proposed framework for developing indicators of tal Protection, Hartford.
ecosystem health. Hydrobiologia 263:1. BATZER, D.P., R.B. RADER & S.A.WISSINGER. 1999. Invertebrates in
GURTZ, M.E. 1994. Design of biological components of the National Freshwater Wetlands of North America: Ecology and Management.
Water-Quality Assessment (NAWQA) Program. In S.L. Loeb & A. John Wiley & Sons, New York, N.Y.
Spacie, eds. Biological Monitoring of Aquatic Systems. Lewis GRIFFITHS, R.W. 1999. BioMAP: Bioassessment of Water Quality. Cen-
Publishers, Ann Arbor, Mich. tre for Environmental Training, Niagara College, Niagara-on-the-
DAVIS, W.S. & T.P. SIMON, eds. 1995. Biological Assessment and Lake, Ontario, Canada.
Criteria: Tools for Water Resource Planning and Decision Making. KARR, J.R. & E.W. CHU. 1999. Restoring Life in Running Waters. Island
Lewis Publishers, Boca Raton, Fla. Press, Washington, D.C.
HAUER, F.R. & G.A. LAMBERTI, eds. 1996. Methods in Stream Ecology.
GERRITSEN, J., B. JESSUP, E.W. LEPPO & J. WHITE. 2000. Development of
Academic Press, New York, N.Y.
lake condition indexes (LCI) for Florida. Tetra Tech, Inc., Owings
KARR, J.R. 1996. Aquatic invertebrates: sentinels of watershed condi-
Mills, Md. Appendices A-1 to B-13, Florida Dep. Environmental
tion. Wings 19(2):22.
U.S. ENVIRONMENTAL PROTECTION AGENCY. 1996. Biological Criteria: Protection, Stormwater & Nonpoint Source Management Section,
Technical Guidance for Streams and Small Rivers. EPA 822-B-96- Tallahassee.

10500 B. Sample Collection

1. General Considerations Characterize the physicochemical properties of faunal sam-

pling station substrate and overlying water. Measure such prop-
Before conducting a benthic survey, determine specific data erties as sediment size class distribution (sand, silt, and clay);
quality objectives (DQOs) and define clearly the information organic content and toxic pollutant concentrations; temperature,
sought. DQOs are qualitative and quantitative statements devel- salinity, hardness, alkalinity, dissolved oxygen, total organic
oped to specify the quality of data needed to support specific carbon (TOC), ammonia, sulfides, and nutrient (total and dis-
decisions and conclusions about the information sought. Discus- solved) concentrations; biochemical oxygen demand; water
sion with water chemists, hydrologists, limnologists, and indi- depth; and velocity of flowing streams.
viduals from other disciplines will be helpful. Ultimate selection After gaining a thorough understanding of the factors involved
of a methodology will depend on whether the habitat to be with a particular body of water, select specific areas to be
studied is a stream, lake, reservoir, or marine area. For example, sampled. There is no set number of sampling stations that will be
to determine whether the macroinvertebrate community down- appropriate to monitor all possible waste discharges. No water
stream from a discharge is damaged, only a few sampling sta- quality survey is routine, nor can one be conducted totally on a
tions upstream and downstream from the discharge are needed. “cookbook” basis. However, if some basic rules such as the
However, if the objective is to delimit the extent of damage from following are adhered to, a sound survey can be designed:
a discharge or series of discharges, it is necessary to have 1. Always establish a reference station(s) upstream or at a
reference stations upstream from all discharges, to bracket each point remote from all wastewater discharges of concern. Because
discharge with stations, and to establish stations downstream. In most surveys are made to determine the damage that pollution
marine waters, it may be necessary to sample a nearby estuary causes to aquatic life, this will be the basis for comparison of the
or, for open ocean waters, to sample some distance from the biota in polluted and unpolluted areas. Preferably have at least
discharge point. two reference stations, one well away from, or upstream from,
10-66 BIOLOGICAL EXAMINATION (10000)

the discharge and the other directly above, or in the immediate 2. Sampling Design
vicinity of, the effluent discharge, but not subject to its influence.
Whenever it is feasible, use reference stations having physico- In biology, the term population refers to a group of individuals
chemical characteristics similar to those of the substrate and that are all members of the same species or taxonomic group. In
overlying water of the receiving area. statistics, population refers to the entire set of values for the
2. Locate a station immediately downstream or in the affected characteristic of interest in a whole sampling universe. For
area in the immediate vicinity of each discharge. example, researchers interested in determining the mean density
3. If the discharge does not mix completely on entering the of worms in the bottom of a lake might take ten grabs from the
body of water, but channels along one side or disperses in a lake sediments. The number of worms in each grab would be an
specific direction, locate stations in the left-bank (looking up- observation, the density of worms would be the characteristic of
interest, and the contents within each grab would be the exper-
stream), midchannel, and right-bank sections of the stream, and
imental unit or sampling unit. The entire bottom of the lake
in concentric arcs in lakes and oceanic waters, or any other
would be the sampling universe and enough grabs to equal the
configuration that will meet study objectives.
area of the entire lake bottom would be the population (of units).
4. Establish stations at various distances downstream from the Similarly, the term sample has two, often contradictory, usages.
last discharge of concern to determine the linear extent of dam- In typical studies, observations usually are not made for all the
age. In the marine environment, an estuary nearby may be possible sampling units. Instead, observations are made that make
sampled or in open ocean waters samples may be taken in a up only a small fraction of the total possible number of observations
nearby area comparable with respect to currents, depth, sediment that could be made. Statistically, this set of observations is referred
characteristics, and salinity. to as a sample. In the example given above the ten grabs would be
5. To permit comparison of macroinvertebrate communities, a sample. However, in everyday language and as used in this book
be sure that all sampling stations are ecologically similar. For and most scientific publications, the term “sample” has been used to
example, select stations that are similar with respect to bottom signify a portion of the real world that has been selected for
substrate (e.g., sand, gravel, rock, mud, organic content), depth, measurement, such as a water sample, plankton haul, or bottom
presence of riffles and pools, stream width, gradient, flow ve- grab. Therefore, each of the grabs in the example above would be
locity, bank or shore cover, salinity, or hardness, TOC, nutrient considered a sample, i.e., “ten samples were taken.”
and dissolved oxygen concentrations, and wave exposure. Collecting a representative sample is difficult because of vari-
6. Collect samples for physical, toxicological (if applicable), ation in successive samples. Without knowledge of sampling
and chemical analyses as close as possible to biological sampling variability, the degree to which the data truly represent the
stations to assure correlation of findings; take such samples at the population cannot be known. Make replicate observations of a
same time and from the same grab when possible. Collect population if definitive statistical inferences about the population
substrate samples for physicochemical analyses from the upper are to be made.2–11
few centimeters where most organisms live. Standardize sampling design to consider the following re-
quirements:
7. Locate sampling stations for macroinvertebrates in the best
1. Approximate the set of all samples that can be selected (i.e.,
physical habitat areas that are not influenced by atypical condi-
separate the sampling universe into all possible samples). For
tions (e.g., bridges, dams, etc.).
example, if the location (site) containing the population has an
8. Discharges in areas near a coast may be subject to variation area of 1000 m2 and the sampling device samples an area of 1
in degree of salt water intrusion (salt water wedge). In such m2, there are 1000 samples that could be collected in the sam-
areas, macroinvertebrate populations may change drastically; pling universe.
document and/or allow for this effect. 2. Assign each sample an equal probability of being selected.
9. When sampling in small, wadeable, first- to third-order Using the situation above, divide the area to be sampled into
streams, initiate sampling at the most downstream station and then 1000 discrete units.
proceed upstream to minimize disruptions induced by the sampling 3. Use a table of random numbers to select sites for sampling,
itself. This is not necessary for nonwadeable streams and rivers. i.e., sample randomly, not haphazardly.
For a long-term biological monitoring program, consider col- 4. The sampling design outlined above is known as simple
lecting macroinvertebrates at each station at least once during random sampling. It is often advantageous to determine the
each of the annual seasons, though this may not always be number of samples necessary for a certain level of precision
necessary and would depend on the study design.1 More frequent while using this type of sampling design. Use the following
sampling may be necessary if the characteristics of the effluents formula to estimate this number:
change or if spills occur. Make allowance for collections at night
where “drift” or night feeding organisms are of special concern.
In general, the most critical period for macroinvertebrates in
N⫽ 冉 冊
t⫻s
D ⫻ x៮
2

streams is during periods of high temperature and low flow,

whereas in estuarine and marine environments it is the period of where:
maximum stratification and poor vertical mixing. If available N ⫽ number of samples,
time and funds limit sampling frequency, make at least one t ⫽ tabulated t value at 0.05 level with the degrees of freedom
survey during the critical time. of preliminary survey (generally t ⬇ 2.0 at larger sam-
pling sizes),
s ⫽ sampling standard deviation of samples, known from a
BENTHIC MACROINVERTEBRATES (10500)/Sample Collection 10-67

preliminary survey,
D ⫽ required level of precision expressed as a decimal (0.30 to
0.35 usually yields a statistically reliable estimate), and
x៮ ⫽ sample mean density of preliminary survey.

Specific information (the mean and standard deviation) about

the population to be sampled is necessary to estimate the number
of samples. Because this information is unknown because sam-
pling has yet to take place, estimate the population’s mean and
standard deviation by one of three ways: conduct a pilot study;
use results from an earlier or a similar study; or make educated
estimates of the population mean and standard deviation.3,12 As
an example, in determining the mean chironomid density in
relatively homogeneous lake sediments during the summer, and
having information that six grabs taken the previous summer
produced a mean density of 4230 chironomids/m2 and a standard Figure 10500:1. Ponar®* grab.
deviation of 1628 chironomids/m2, it may be satisfactory if the
final estimate of mean chironomid density is correct within ⫾
30%, with a 5% probability of error (␣ ⫽ 0.05). Using the most common quantitative sampling devices are the Ponar®*, Pe-
formula given above, tersen, and Ekman grabs and the Surber or square-foot stream

N⫽ 冉 2.5706 ⫻ 1628
0.30 ⫻ 4230
冊 2 bottom sampler, all described below.
a. Grab samplers:
Measure each grab-type sampler for actual surface area sampled
(t ⫽ 2.5706 at a 5% probability of error and 5 degrees of freedom) before it is first used.
1) The Ponar®* grab (Figure 10500:1) is used increasingly in
N ⫽ 10.88 ⬇ 11 medium to deep rivers, lakes, reservoirs, and estuaries.14 It is similar
to the Petersen grab in size, weight, lever system, and sample
Thus it is estimated that 11 grabs will be necessary. compartment, but has side plates and a screen on top of the sample
5. Simple random sampling is useful in sampling relatively ho- compartment to prevent sample loss during closure. With one set of
mogeneous areas. However, most taxa are not distributed uniformly weights, the standard 23- by 23-cm sampler weighs 20 kg. A 15- by
over water bottoms. Different habitats (sand, mud, gravel, or or- 15-cm petite Ponar®* may be used. The large surface disturbance
ganic material) support different densities and species of organisms. associated with a Ponar®* grab can be reduced by installing hinged,
In such circumstances, use a stratified random design. In this sam- rather than fixed, screen tops, thereby reducing the pressure wave
pling design a heterogeneous universe (different bottom substrates, associated with the sampler’s descent. This sampler is best used for
current velocities, depths, temperatures, etc.) is divided into more mud, sand, gravel, or small rocks with mud, but it can be used in all
homogeneous strata. Once the strata are defined, use random sam- substrates except bedrock.
pling, as above, within each stratum. Stratified random sampling has 2) The Petersen grab (Figure 10500:2) is used for sampling hard
two important advantages: it is often valuable to have data on the bottoms such as sand, gravel, marl, and clay in swift currents and
various subsets of a population (e.g., density of benthic inverte- deep water.3 It is an iron, clam-type grab manufactured in various
brates in each of the sediment types), and stratified sampling often sizes that will sample an area of from 0.06 to 0.09 m.2 It weighs
reduces variability because it deals with more homogeneous sub- approximately 13.7 kg, but may weigh as much as 31.8 kg when
populations, allowing for more accurate (closer to the actual value)
and precise (less variation among the values) population estimates.
Prior information is necessary to divide the population into the * Registered trademark of Morris & Lee, Inc. d/b/a Wildlife Supply Co., Buffalo,
various strata. This is usually accomplished through pre-study re- NY.
connaissance (a pilot study). Systematic sampling, a third type of
sampling design (in addition to simple random and stratified ran-
dom), often is used in such pilot studies. In a systematic-transect
design, conduct sampling at equal intervals along a number of
transects within a habitat. This design can be used to identify and
locate the existent strata.3,12
6. In descriptive studies investigators should take at least three
replicate sampling units per station.3,13 If statistical testing is
planned, more replicates probably are necessary.
7. Standardize data acquisition and recording when practical. Use
metric units.
3. Sampling Devices, Quantitative

Quantitative and qualitative samplers have been designed to

collect organisms from the bottom of different water bodies. The Figure 10500:2. Petersen grab.
10-68 BIOLOGICAL EXAMINATION (10000)

Figure 10500:3. Van Veen grab.

auxiliary weights are bolted to its sides. The primary advantage of

the extra weights is to make the grab stable in swift currents and to Figure 10500:5. Shipek®* grab.
give additional cutting force in fibrous or firm bottom materials.
Modify the sampler by adding end plates, by cutting large strips out
at the top of each side, and by adding a hinged 30-mesh screen as is composed of two concentric half cylinders. When the grab
in the Ponar®* grab.15 touches bottom, inertia from a self-contained weight releases a catch
To use the Petersen grab, set the hinged jaws and lower to the and helical springs rotate the inner half cylinder by 180°. The
bottom slowly to avoid disturbing lighter bottom materials. Ease sample bucket may be disengaged from the upper semi-cylinder by
rope tension to release the catch. As the grab is raised the lever releasing two retaining latches. This grab is for special use in marine
system closes the jaws. waters and large inland bodies of water, for instance, in compact
3) The Van Veen grab (Figure 10500:3) is used to sample in substrates.
open marine waters and in large lake environments. The long arms 6) The Ekman grab (Figure 10500:6) is useful only for sampling
of the sampler tend to act as stabilizers without disturbing the water mud, silt, muck, and sludge in water with little current.3 It is difficult
at the water-substrate interface. It is basically an improved version to use when rocky or sandy bottoms or moderate macrophyte
of the Petersen grab for mud, gravel, pebble, and sand substrates. growth are present because small pebbles or grit or macrophyte
The sampler is heavy; lower it from a boat or ship platform with stems prevent proper jaw closure. The grab weighs approximately
mechanical or hydraulic lifts. 3.2 kg. The box-like part holding the sample has spring-operated
4) The Smith-McIntyre grab (Figure 10500:4) has the heavy steel
construction of the Petersen, but its jaws are closed by strong coil
springs.16 Chief advantages are its stability and easier control in
rough water. Its bulk and heavy weight require operation from a
large boat equipped with a winch. The 45.4-kg grab can sample an
area of 0.2 m2,17–19 but smaller models (0.1 m2 or 0.05 to 0.06 m2)
are available.
5) The Shipek®* grab (Figure 10500:5) is designed to take a
sample 0.04 m2 in surface area and approximately 10 cm deep at the
center in virtually any type of substrate.3 The sample compartment

* Registered trademark of Morris & Lee, Inc. d/b/a Wildlife Supply Co., Buffalo,
NY.

Figure 10500:4. Smith-McIntyre grab. Figure 10500:6. Ekman grab.

BENTHIC MACROINVERTEBRATES (10500)/Sample Collection 10-69

A common problem in using the Surber sampler is that organisms

wash under the bottom edge of the sampler. The following modi-
fications have been suggested for different substrates:
For loose gravel—Extend bottom edge of Surber frame to 5 or
more cm allowing for insertion of frame into substrate to a greater
depth. This method works well in soft substrates such as sand and
gravel where the current causes substrate shifting.
For coarse gravel and rock—Add serrated extension to back edge
of frame to secure it and reduce washing from under this edge. This
Figure 10500:7. Surber or square-foot sampler. method is helpful in hard gravel and rock substrates where sinking
the entire frame is impossible.
jaws on the bottom that must be cocked manually (exercise caution For gravel and bedrock—Add a 5-cm band of flexible material to
in cocking and handling the grab because of possible injuries if jaws bottom edge of sampler to create a seal in rocky, uneven substrates.
are tripped accidentally). At the top of the grab are two hinged Make band of foam rubber or fine-textured synthetic sponge. Re-
overlapping lids that are held open partially during descent by water move organisms that stick to foam and include in sample.
passing through the sample compartment. These lids are held shut 2) Hess-type samplers are cylindrical with enclosed sides and an
by water pressure when the sampler is being retrieved. The grab is open top. They function similarly to the Surber-type samplers.3
made in three sizes: 15 ⫻ 15 cm, 23 ⫻ 23 cm, and 30 ⫻ 30 cm, but c. Core or cylindrical samplers: Use core or cylindrical samplers
the smallest size usually is adequate. A taller model of this sampler, to sample sediments in depth. Efficient use as surface samplers
either 23 cm or 30.5 cm tall, is available. To prevent sample requires dense animal populations. Core samplers vary from hand-
overflow and loss, place a Standard U.S. No. 30 sieve insert in the pushed tubes to explosive-driven and automatic-surfacing mod-
top for deep sediments. els.3,21
b. Riffle/run samplers: 1) The Phleger corer (Figure 10500:8) is widely used. It operates
1) Surber-type samplers (Figure 10500:7)20 consist of two brass on the gravity principle.3 Styles and weights vary among manufac-
frames, each 30.5 cm (1 ft) square, hinged together along one edge. turers; some use interchangeable weights that allow variations be-
When in use, the two frames are locked at right angles, one frame tween 7.7 and 35.0 kg, while others use fixed weights weighing 41.0
marking off the area of substrate to be sampled, and the other kg or more. Length of core taken will vary with substrate texture.
supporting a net to collect organisms washed into it from the sample
area.
The net usually is 69 cm long with the first few centimeters and
the wings constructed of heavier material (canvas, taffeta) to in-
crease durability. Standard 30 mesh size is 595 to 600 ␮m. While a
smaller mesh size might increase the number of smaller inverte-
brates and young instars collected, it also will clog more easily and
exert more resistance to the current than a larger mesh. This could
result in a loss of organisms due to backwashing from the sample
net. This sampler is specific for macrobenthos; many microcompo-
nents of the benthos are not collected.
Use this sampler in shallow (30 cm or less), flowing water. When
it is used in deeper water some organisms may be carried over the
top of the sampler. Position sampler securely on the stream bottom
parallel to water flow with the net portion downstream. Take care
not to disturb the substrate upstream from sampler. Leave no gaps
under the edges of the frame that would allow water to wash under
the net. Fill gaps that may occur along the back edge of the sampler
by carefully shifting rocks and gravel along the outside edge. When
the sampler is in place (it may be necessary to hold it in place with
one hand in a strong current), carefully turn over and lightly hand-
rub all rocks and large stones inside the frame to dislodge organisms
clinging to them. Examine each stone for organisms, larval or pupal
cases, etc., that may be clinging to it before discarding. Scrape
attached algae, insect cases, etc., from the stones into the sampler
net. Stir remaining gravel and sand with the hands or a stick to a
depth of 5 to 10 cm, depending on the substrate, to dislodge
bottom-dwelling organisms. It may be necessary to hand-pick some
mussels and snails that are not carried into the net by the current.
Remove sample by inverting net into sample container. Carefully
examine net for small organisms clinging to it. Remove these,
preferably with forceps to avoid damage, and include in sample. Figure 10500:8. Phleger core sampler.
Rinse sampler net after each use.
10-70 BIOLOGICAL EXAMINATION (10000)

2) The KB®* core sampler (Figure 10500:9) or a modification

known as the Kajak-Brinkhurst corer, may be useful in obtaining
estimates of the standing stock of benthic macroinvertebrates in-
habiting soft sediments.22
3) Box core samplers23–26 can sample a variety of sediment
types. They are available in several sizes and are used in marine
waters and in the Great Lakes3,27 to collect benthic macrofauna.
These devices may be deployed from ships or other platforms or
they may be used by divers. Preferably use a box coring device with
a rectangular corer, having a cutting arm that can seal the sample
before retraction from the bottom. To sample a sufficient number of
individuals and taxa, and to integrate the patchy distribution of the
benthic fauna, use a sampler with a surface area of no less than 100
cm2 and a sediment depth of at least 20 cm. A box corer capable of
sampling deeper sediment may be needed to collect deep burrowing
infauna. In sandy-type sediments, it may be necessary to substitute
a grab sampler for the box corer to achieve adequate sediment
penetration and collection. Visually inspect each sample to ensure Figure 10500:10. Wilding or stovepipe sampler.
that an undisturbed and adequate amount of sample is collected.
4) The Wilding or stovepipe sampler (Figure 10500:10)28 is
made in various sizes and with many modifications.3 It is especially
useful for quantitatively sampling a bottom with dense, vascular d. Drift samplers: Drift samplers, usually in the form of nets
plant growth. It may be used to sample vegetation, mud-water (Figure 10500:11) , are anchored in flowing water for capture of
interface sediment, or most shallow stream substrates. However, macroinvertebrates that have migrated or have been dislodged from
large volumes of vegetation, when sampled in this way, may require the bottom substrates into the current. Drift organisms are important
a great deal of time for laboratory processing. to the stream ecosystem because they are prey for stream fish and
should be considered in the study of fish populations. Drift organ-
isms respond to pollutional stresses, including spills, by increased
drift from an affected area; therefore, drift is important in water-
* Registered trademark of Morris & Lee, Inc. d/b/a Wildlife Supply Co., Buffalo,
NY. quality investigations, especially of spills of toxic materials. Drift
also is a factor in recolonizing denuded areas and it contributes to
recovery of disturbed streams.
Use nets having a 929-cm2 upstream opening and mesh equiva-
lent to U.S. Standard No. 30 screen (595-␮m pore size). After
placing the net in the water, frequently remove organisms and
debris to prevent clogging and subsequent diversion of water at the
net opening. Use replicate samples as appropriate to meet study
objectives. Set drift-net samples for any specified time (usually 1 to
3 h) but use the same time for each station. Sampling between dusk
and 1 AM is optimum.
The total quantity (numbers or biomass) of organisms drifting
past a given station is the best measure of drift intensity. Report data
in terms of numbers or biomass/m3.2,29 –31

4. Sampling Devices, Qualitative

When sampling qualitatively, search for organisms in as many

different habitats as possible.32 Collect samples by any method
that will capture representative species.

Figure 10500:9. KB®* corer. Figure 10500:11. Drift net sampler.

BENTHIC MACROINVERTEBRATES (10500)/Sample Collection 10-71

Placement conditions should be similar, so the numbers and

kinds of organisms reflect capacity to support aquatic life.
Position artificial substrates in the euphotic zone (0.3 m) for
maximum abundance and diversity of macroinvertebrates.13 Op-
timum time for substrate colonization is 6 weeks for most waters.
For uniformity of depth, suspend sampler from floats on a
3.2-mm steel cable. If vandalism is a problem, use subsurface
floats or place sampler near the bottom. Regardless of installa-
tion technique, use uniform procedures.
At shallow water stations (less than 1.2 m deep), install
samplers so that they are located midway in the water column at
low flow. For samplers installed in July when the water depth is
about 1.2 m and the August average low flow is 0.6 m, install
0.3 m above the bottom. Take care not to let samplers touch the
bottom or they may become covered with silt, thereby increasing
the sampling error. In shallow streams with sheet rock bottoms,
secure artificial substrates to 0.95-cm steel rods that are driven
into the substrate or secure to rods that are mounted on low, flat,
rectangular blocks.
Before removing samples from the water, it may be necessary
to enclose them in an oversized plastic bag (double wrapping)
that is tightly sealed to prevent possible loss of organisms or to
use a large dip net (openings equivalent to a U.S. Standard No.
30 sieve) when the sample is removed. Disassemble sampler and
brush in a pan of water in the field or add preservative to the bag
containing the intact sampler, and disassemble and brush later in
the laboratory.
Although many different styles of artificial substrate samplers
have been tested,37 the Fullner38 modification of the Hester-
Dendy39 multiplate and the basket sampler13 are used widely.
a. Multiple-plate or modified Hester-Dendy sampler (Figure
Figure 10500:12. Hester-Dendy artificial substrate unit. 10500:12) is constructed of 0.3-cm-thick tempered hardboard
with 7.5-cm round plates and 2.5-cm round spacers that have
center-drilled holes. The plates are separated by spacers on a
a. Dip, kick nets are the most versatile collecting devices for 0.63-cm-diam eyebolt, held in place by a nut at the top and
shallow, flowing water, and are useful also for shoreline collect- bottom. In each sampler, 14 large plates and 24 spacers are used.
ing in lakes. When combined with a standardized kicking tech- Separate the top 9 plates by a single spacer. Separate Plate 10 by
nique,33 these nets are appropriate for quantitatively sampling two spacers, Plates 11 and 12 by three spacers, and Plates 13 and
macroinvertebrates.34 14 by four spacers. The sampler is approximately 14 cm long and
b. Tow nets, dredges, or trawls range from simple sled- 7.5 cm in diameter, has an exposed surface area of approxi-
mounted nets to complicated devices incorporating teeth that dig mately 1300 cm2, and weighs about 0.45 kg. Do not reuse
into the bottom. Some models feature special apparatus to hold samplers exposed to oils and chemicals that may inhibit coloni-
the net open during towing and to close it during descent and zation. Because it is cylindrical, the sampler fits a wide-mouth
retrieval. Available styles have been discussed elsewhere.21,35,36 container for shipping and storage. The sampler is inexpensive,
compact, and lightweight.13,38,39
5. Sampling Devices, Artificial Substrate Samplers A different type of square modified Hester-Dendy, multiple-
plate artificial substrate sampler is constructed of 0.3-cm tem-
Artificial substrate samplers are devices of standard composi- pered hardboard cut into 7.6-cm square plates and 2.5-cm square
tion and configuration placed in the water for a predetermined spacers.32 Eight plates and twelve spacers are used for each
exposure period for colonization by macroinvertebrate commu- sampler. The plates and spacers are placed on a 1/4-in. (0.64-cm)
nities. Because many physical variables encountered in bottom
sampling are minimized, e.g., depth, light penetration, tempera-
ture differences, and species substrate preferences, artificial sub-
strate sampling complements other types of sampling. Like nat-
ural submerged substrates such as logs and pilings, artificial
substrates are colonized primarily by immature aquatic insects,
crustaceans, coelenterates, bryozoans, and to some extent
worms, gastropods, and mollusks. In lotic systems the organisms
that colonize artificial substrates are primarily drift organisms,
such as immature insects and eggs, carried by water currents. Figure 10500:13. Basket sampler.
10-72 BIOLOGICAL EXAMINATION (10000)

Figure 10500:14. Marsh net sampler.

eyebolt so that there are three single spaces, three double spaces, rectly on specific sampling sites, but a SCUBA diver is required
and one triple space between the plates. The total surface area of to collect samples.43 Improved accuracy of locating sampling
the sampler, excluding the eyebolt, is 939 cm2 (0.9 m2). Gener- sites and ability to collect a large number of replicate samples
ally, five samplers are used and placed in streams tied to a may outweigh the disadvantage of using a diver. Suction sam-
concrete construction block as anchor. This prevents samplers plers have been used widely in sampling marine environments,
from coming into contact with the natural substrates. but they have obvious depth limitations.
b. The basket sampler13 (Figure 10500:13) is a cylindrical
“barbecue” basket 28 cm long and 17.8 cm in diameter, filled
with approximately 30 5.1-cm-diam rocks or rocklike material 7. References
weighing 7.7 kg. A hinged side door allows access to the
contents. The sampler provides an estimated 0.24 m2 of surface 1. ALDEN, R.W., III, S.B. WEISBERG, J.A. RANASINGHE & D.M. DAUER.
area for colonization. The factors governing proper installation 1997. Optimizing temporal sampling strategies for benthic environ-
and collection are the same as those described for the multiplate mental monitoring programs. Mar. Pollut. Bull. 34:913.
sampler. Some investigators prefer using the basket because 2. SNEDECOR, G.W. & W.G. COCHRAN. 1967. Statistical Methods. Iowa
natural substrate materials are used for colonization. State Univ. Press, Ames.
c. Marsh net sampler (Figure 10500:14) is used for sampling 3. KLEMM, D.J., P.A. LEWIS, F. FULK & J.M. LAZORCHAK. 1990. Mac-
macroinvertebrates in estuarine and marine environments.40 It roinvertebrate Field and Laboratory Methods for Evaluating the
can be used in different habitats (e.g., marsh, beach, tidal creek, Biological Integrity of Surface Waters. EPA-600/4-90-030. Envi-
and tidal flat) of estuarine and marine intertidal zones to depths ronmental Monitoring Systems Laboratory, U.S. Environmental
of 3 m. The metal frame is constructed of No. 22 galvanized Protection Agency, Cincinnati, Ohio.
sheet metal and 1/4-in. (6-mm) welding rods. A 0.5-m plankton 4. GREEN, R.H. 1979. Sampling Design and Statistical Methods for
net of nylon monofilament screen is laced to the posterior end of Environmental Biologists. John Wiley & Sons, New York, N.Y.
the frame. The net has a bayonet-type cod end for easy removal. 5. ZAR, J.H. 1984. Biostatistical Analysis, 2nd ed. Prentice-Hall,
The mesh size of the plankton net and cod end is about 1 mm Englewood Cliffs, N.J.
(bar measure). The frame and net weigh 5 kg. The collecting 6. UNDERWOOD, A.J. & C.H. PETERSON. 1988. Towards an ecological
framework for investigating pollution. Mar. Ecol. Progr. Ser. 46:227.
procedures are the same in all habitats of the intertidal zone. The
7. MANTEL, N. 1987. The detection of disease clustering and a gener-
net is placed at one end of the sampling area and 30 m of rope
alized regression approach. Cancer Res. 27:200.
is paid out in an arc to prevent the operator from disturbing the
8. LEGENDRE, P. & M.J. FORTIN. 1989. Spatial patterns and ecological
sampling site. The net is then retrieved by hand at a rate of about analysis. Vegetation 80:107.
0.3 m/s. Advantages are that the sampling distance does not have 9. GREEN, R.H., J.M. BOYD & J.S. MACDONALD. 1993. Relating sets of
to be measured before taking the sample, the net can be towed at variables in environmental studies: the sediment quality triad as a
a constant speed, and samples also can be taken over soft mud paradigm. Environmetrics 4:439.
bottoms.40 10. UNDERWOOD, A.J. 1994. On beyond BACI: sampling designs that
might reliably detect environmental disturbances. Ecol. Appl. 4:3.
6. Suction Samplers 11. LANDIS, W.G., G.B. MATTHEWS, R.A. MATTHEWS & A. SARGENT.
1994. Application of multivariate techniques to endpoint determi-
Suction samplers are used widely for collecting benthic mac- nation, selection and evaluation in ecological risk assessment. En-
roinvertebrate samples.41,42 These samplers can be placed di- viron. Toxicol. Chem. 13:1917.
BENTHIC MACROINVERTEBRATES (10500)/Sample Collection 10-73

12. GAUGUSH, R.F. 1987. Sampling Design for Reservoir Water Quality 33. FROST, S., A. HUN & W. KERSHAW. 1971. Evaluation of a kicking
Investigations. Instruction Rep. E-87-1, Waterways Experiment Sta- technique for sampling stream bottom fauna. Can. J. Zool. 49:167.
tion, Vicksburg, Miss. National Technical Information Serv., 34. CROSSMAN, J.S., J. CAIRNS, JR. & R.L. KAESLER. 1973. Aquatic
Springfield, Va. Invertebrate Recovery in the Clinch River Following Hazardous
13. MASON, W.T., JR., C.I. WEBER, P.A. LEWIS & E.C. JULIAN. 1973. Spills and Floods. Water Resour. Res. Center Bull. 63, Virginia
Factors affecting the performance of basket and multiplate macro- Polytechnic Inst. & State Univ., Blacksburg.
invertebrate samplers. Freshwater Biol. 3:409. 35. WELCH, P.S. 1948. Limnological Methods. Blakiston Co., Philadel-
14. POWERS, C.F. & A. ROBERTSON. 1967. Design and Evaluation of an phia, Pa.
All-Purpose Benthos Sampler. Spec. Rep. No. 30, Great Lakes 36. USINGER, R.L. 1956. Aquatic Insects of California, with Keys to
Research Div., Univ. Michigan, Ann Arbor. North American Genera and California Species. Univ. California
15. WEBER, C.I., ed. 1973. Biological Field and Laboratory Methods for Press, Berkeley.
Measuring the Quality of Surface Waters and Effluents. EPA-670/ 37. BEAK, T.W., T.C. GRIFFING & G. APPLEBY. 1974. Use of artificial
4-73-001, U.S. Environmental Protection Agency, Cincinnati, Ohio. substrates to assess water pollution. In Proceedings Biological
16. SMITH, W. & A.D. MCINTYRE. 1954. A spring-loaded bottom sam- Methods for the Assessment of Water Quality. American Soc.
Testing & Materials, Philadelphia, Pa.
pler. ]. Mar. Biol. Assoc. U.K. 33:257.
38. FULLNER, R.W. 1971. A comparison of macroinvertebrates collected
17. ELLIOTT, J.M. 1971. Some Methods for the Statistical Analysis of
by basket and modified multiple-plate samplers. J. Water Pollut.
Samples of Benthic Invertebrates. Sci. Publ. No. 25, Freshwater
Control Fed. 43:494.
Biological Assoc., Ambleside, U.K.
39. HESTER, F.E. & J.B. DENDY. 1962. A multiple-plate sampler for
18. MCINTYRE, A.D. 1971. Efficiency of Marine Bottom Samplers. In
aquatic macroinvertebrates. Trans. Amer. Fish. Soc. 91:420.
N.A. Holme & A.D. McIntyre, eds. Methods for the Study of 40. PULLEN, E.J., C.R. MOCK & R.D. RINGO. 1968. A net for sampling
Marine Benthos. IBP Handbook No. 16, p. 140. Blackwell Scientific the intertidal zone of an estuary. Limnol. Oceanogr. 13:200.
Publications, Oxford, England. 41. GALE, W. & J. THOMPSON. 1975. A suction sampler for quantitatively
19. WIGLEY, R.L. 1967. Comparative efficiency of Van Veen and sampling benthos on rocky substrates in rivers. Trans. Amer. Fish.
Smith-McIntyre grab samplers as recorded by motion pictures. Soc. 104:398.
Ecology 48:168. 42. LARSEN, P.F. 1974. A remotely operated shallow water benthic
20. SURBER, E. 1937. Rainbow trout and bottom fauna production in one suction sampler. Chesapeake Sci. 15:176.
mile of stream. Trans. Amer. Fish. Soc. 66:193. 43. SIMMONS, G.M., JR. 1977. The Use of Underwater Equipment in
21. BARNES, H. 1959. Oceanographic and Marine Biology. George Freshwater Research. VPI-SG-77-03, Virginia Polytechnic Inst. &
Allen and Unwin, Ltd., London, England. State Univ., Blacksburg.
22. BRINKHURST, R.O., K.E. CHUA & E. BATOOSINGH. 1969. Modifica-
tions in sampling procedures as applied to studies on the bacteria
and tubificid oligochaetes inhabiting aquatic sediments. J. Fish Res. 8. Bibliography
Board Can. 26:2581.
23. HOLME, N.A. & A.D. MCINTYRE, eds. 1984. Methods for the Study MACAN, T.T. 1958. Methods of sampling bottom fauna in stony streams.
of Marine Benthos. IBP Handbook 16, Blackwell Scientific Publ., Mitt. Int. Ver. Limnol. 8:1.
Oxford, England. DICKSON, K.L., J. CAIRNS, JR. & J.C. ARNOLD. 1971. An evaluation of the
24. HESSLER, R.R. & P.A. JUMARS. 1974. Abyssal community analysis from use of a basket type artificial substrate for sampling macroinverte-
replicate box cores in the central north Pacific. Deep-Sea Res. 21:185. brate organisms. Trans. Amer. Fish. Soc. 100:553.
25. PROBERT, P.K. 1984. A comparison of macrofaunal samples taken WASHINGTON, H.G. 1984. Diversity, biotic and similarity indices. A
by box corer and anchor-box dredge. New Zealand Oceanogr. Inst. review with special relevance to aquatic ecosystems. Water Res.
Rec. 4(13):149. 18:653.
26. ELEFTERIOU, A. & N.A. HOLME. 1984. Macrofauna techniques. In WRONA, F.J., P. CALOW, T. FORD, D.J. BAIRD & L. MALTBY. 1986.
N.A. Holme & A.D. McIntyre, eds. Methods for the Study of Estimating the abundance of stone-dwelling organisms: a new
Marine Benthos. IBP Handbook 16, Chapter 6, p. 140. Blackwell method. Can. J. Fish. Aquat. Sci. 43:2025.
Scientific Publ., Oxford, England. BRITTAIN, J.E. & T.J. EIKELAND. 1988. Invertebrate drift—A review.
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27. REYNOLDSON, T.B. & A.L. HAMILTON. 1993. Historic changes in
FERRARO, S.P., F.A. COLE, W.A. DEBEN & R.C. SWARTZ. 1989. Power-
populations of burrowing mayflies (Hexagenia limbata) from Lake
cost efficiency of eight macroinvertebrate sampling schemes in
Erie based on sediment tusk profiles. J. Great Lakes Res. 19:250.
Puget Sound, Washington, U.S.A. Can. J. Fish. Aquat. Sci. 46:
28. WILDING, J.L. 1940. A new square-foot aquatic sampler. Limnol.
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Soc. Amer. Special Publ. No. 4, Ann Arbor, Mich.
SCRIMGEOUR, G.J., J.M. CULP & N.E. GLOZIER. 1993. An improved
29. WATERS, T.F. 1961. Standing crop and drift of stream bottom technique for sampling lotic invertebrates. Hydrobiologia 254:65.
organisms. Ecology 42:532. CUFFNEY, T.F., M.E. GURTZ & M.R. MEADOR. 1993. Methods for col-
30. DIMOND, J.B. 1967. Pesticides and Stream Insects. Bull. No. 2, lecting benthic invertebrate samples as part of the National Water-
Maine Forest Serv., Augusta, & Conservation Foundation, Wash- Quality Assessment Program. Open-File Rep. 93-405, U.S. Geo-
ington, D.C. logical Survey, Raleigh, N.C.
31. WATERS, T.F. 1972. The drift of stream insects. Annu. Rev. Entomol. HUGHES, R.M. 1995. Defining acceptable biological status by comparing
17:253. with reference conditions. In W.S. Davis and T. Simon, eds. Bio-
32. OHIO ENVIRONMENTAL PROTECTION AGENCY. 1989. Biological Criteria logical Assessment and Criteria, Tools for Water Resource Plan-
for the Protection of Aquatic Life: Volume II, Users Manual for ning and Decision Making, Chapter 4, p. 31. Lewis Publishers,
Biological Field Assessment of Ohio Surface Waters & Volume III, Boca Raton, Fla.
Standardized Biological Field Sampling and Laboratory Methods REYNOLDSON, T.B., R.H. NORRIS, V.H. RESH, K.E. DAY & D.M. ROSEN-
for Assessing Fish and Macroinvertebrates Communities. Ohio En- BURG. 1997. The reference condition: a comparison of multimetric
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10-74 BIOLOGICAL EXAMINATION (10000)

HEINO, J. 2000. Lentic macroinvertebrate assemblage structure along mental Fate; Biotechnology; Pesticides, Standards D-4342-84,
gradients in spatial heterogeneity, habitat size and water chemistry. D-4343-84, D-4344-84, D-4345-84, D-4346-84, D-4347-84,
Hydrobiologia 428:229. D-4348-84, D-4387-84, D-4401-84, D-4407-84, D-4556-85,
AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2000. Annual Book of D-4557-85, D-4558-85. American Soc. Testing & Materials, W.
Standards. Section 11, Vol. 11.05, Biological Effects and Environ- Conshohocken, Pa.

10500 C. Sample Processing and Analysis

1. Sample Processing For qualitative samples, place rocks, sticks, and other objects
in a white pan partially filled with water. Many animals will float
After collecting a benthic sample, transfer it to either specially free from these objects and can be removed with forceps.
designed sieve tables (or hoppers) or a container. If a container Assign identification numbers either in the field or at the
(such as small trash can) is used, dilute with ambient water and laboratory and transcribe information from the labels to a per-
swirl. Pour slurry gradually into a sieve bucket. Gently wash manent ledger. The ledger provides a convenient reference in
slurry over screen to prevent damaging or losing specimens. identifying number of samples collected at various places, time
Slurries that clog the screen require removal of screened mate- of sampling, and water characteristics.
rial. A series of one or two coarser screens (e.g., 1-cm and Preserve and store in 70% ethanol organisms taken in the field
0.5-cm mesh) will hold back larger materials such as leaves, or from artificial substrates and sieved with a U.S. Standard No.
sticks, shells, and gravel while permitting organisms and smaller 30 sieve. For special studies and to retain anatomical form and
materials to pass through to the bottom sieve. Carefully check structures, fix soft-bodied organisms first with 5 to 10% buffered
rocks, sticks, shells, and other objects for attached or burrowed formalin or 70% ethanol. NOTE: For health and safety reasons,
organisms before discarding. A soft-bristled toothbrush may be always take care when using 5 to 10% buffered formalin, or
used to remove attached invertebrates from rocks, sticks, and avoid using it to fix or preserve organisms in the field or in the
similar objects. laboratory. Never discard fixatives or preservatives into the
Wash residual material on the screen into a container. A environment.
cheesecloth bag is very useful because it does not restrict the
quantity of wash water. Label containers with a collection code
but do not affix labels to lids. Similar labels can be written with 2. Sorting and Identification
pencil or indelible ink on high-rag-content paper and placed in
the container. Record label code on a field sheet that describes Whether organisms are sorted in the field or the laboratory,
location, date, type of sample, collector’s name, and other per- follow consistent procedures. Before processing a sample, trans-
tinent information. fer information from the label to a data sheet that provides space
Use laboratory elutriation devices1,2 as appropriate to reduce for scientific names and number of individuals. Place sample
time required to sort benthic organisms from samples containing directly in a shallow white tray with water for sorting. To
large amounts of silt, mud, or clay. Wash screened material into facilitate sorting organisms from detritus, the organisms may be
a container and fix the contents in a solution of 10% buffered stained with rose bengal (200 mg/L or achieve a light pink color)
formalin or 70% ethanol.3– 6 If ethanol is used, do not fill more in the formalin or ethanol preservative for at least 24 h.10 NOTE:
than one-half the container with screened material. Preserve and Excessive staining may prevent specific identification of some
store animals with calcareous shells or exoskeletons, i.e., mus- specimens. Examine entire sample and separate organisms un-
sels, snails, crayfish, and ostracods, in 70% ethanol.6,7 less they occur in very large numbers. If a subsample is sorted,
Some macroinvertebrates (soft-bodied animals) are identified take care that rare forms are not excluded. As organisms are
more easily if they are relaxed to prevent constriction during picked from the sample, sort under a scanning lens or stereo-
preservation. Common relaxants include carbonated water (soda scopic microscope, separate them into different taxonomic cat-
water) or carbon dioxide added to water. Other relaxants include egories, identify to the lowest taxonomic level to meet the data
aqueous solutions of 70% ethyl alcohol, 2% nicotine sulfate, quality objectives, and record on the data sheet. Place animals in
propylene phenoxetol, or 5 to 10% solutions of either chlorotone, separate vials according to category and fill vials with 70%
chloral hydrate, or magnesium sulfate added gradually to the ethanol. Place inside vials labels containing sample tracking
water containing the soft-bodied animals until the degree of number, date collected, sampling location, and names of organ-
relaxation sought is reached. Narcotize organisms before fixing isms.
them. Ideally, fix annelid specimens (oligochaetes) in 5 to 10% Identify animals in each vial using stereoscopic and compound
buffered formalin before preserving them in 70 to 80% ethanol microscopes, according to need, and available experience and
(note that alcohol is not a satisfactory tissue fixative). Fixation resources. Identify organisms to species level if possible. Addi-
stabilizes tissue proteins to retain characteristics of the soft body tional sources of information on laboratory techniques and iden-
(e.g., segmented worms) form.8,9 tification guides and taxonomic keys of macroinvertebrates are
available (see Bibliography and Section 10900).
BENTHIC MACROINVERTEBRATES (10500)/Sample Processing and Analysis 10-75

3. References WILLIAMS, A.B. 1974. Marine flora and fauna of the northeastern United
States. Crustacean: Decapoda. U.S. Circ. No. 389, National Oceanic
1. WORSWICK, J.M. & M.T. BARBOUR. 1974. An elutriation apparatus Atmospheric Admin., National Marine Fisheries Serv., Washing-
for macroinvertebrates. Limnol. Oceanogr. 19:538. ton, D.C.
2. LAUFF, G.H., K.W. CUMMINS, C.H. ERIKSON & M. PARKER. 1961. A FOX, R.S. & K.H. BYNUM. 1975. The amphipod crustaceans of North
method for sorting bottom fauna samples by elutriation. Limnol. Carolina estuarine waters. Chesapeake Sci. 16:223.
Oceanogr. 6:462. MORRIS, P.A. 1975. A Field Guide to Shells of the Atlantic and Gulf
3. EDMONDSON, W.T., ed. 1959. Ward and Whipple’s Freshwater Bi- Coasts and the West Indies. Houghton Mifflin Co., Boston, Mass.
ology, 2nd ed. John Wiley & Sons, New York, N.Y. SMITH, R.I. & J.T. CARLTON, eds. 1975. Light’s Manual: Intertidal
4. COOK, D.G. & R.O. BRINKHURST. 1973. Marine Flora and Fauna of Invertebrates of the Central California Coast, 3rd ed. University of
the Northeastern United States, Annelida: Oligochaeta. NOAA California Press, Berkeley.
Tech. Rep. NMFS CIRC-374, U.S. Dep. Commerce, National Oce- BUTLER, T.H. 1980. Shrimps of the Pacific Coast of Canada. Can. Bull.
anic Atmospheric Admin., National Marine Fisheries Serv., Seattle, Fish. Aquat. Sci. 202:1.
Wash. BLAXTER, J.H.S., S.F.S. RUSSELL & S.M. YONGE. 1980. The species of
5. KLEMM, D.J. 1982. Leeches (Annelida Hirudinea:) of North Amer-
mysids and key to genera. Advan. Mar. Biol. 18:7.
ica. EPA-600/3-82-025, Environmental Monitoring & Support Lab.,
SIEG, J. & R.N. WINN. 1981. The Tanaidae (Crustacea: Tanaidacea) of
U.S. Environmental Protection Agency, Cincinnati, Ohio.
California, with a key to the world genera. Proc. Biol. Soc. Wash.
6. PENNAK, R.W. 1989. Freshwater Invertebrates of the United
States—Protozoa to Mollusca, 3rd ed. John Wiley & Sons, Inc., 94(2):315.
New York, N.Y. HEARD, R.W. 1982. Guide to common tidal marsh invertebrates of the
7. BURCH, J.B. 1972. Freshwater Sphaeriacean Clams (Mollusca: Pe- Northeastern Gulf of Mexico. Alabama Sea Grant Consortium.
lecypoda) of North America. U.S. Environmental Protection MASGP-79-004.
Agency, Cincinnati, Ohio. PRICE, W.W. 1982. Key to the shallow water Mysidacea of the Texas
8. KLEMM, D.J., ed. 1985. A Guide to the Freshwater Annelida coast with notes on their ecology. Hydrobiologia 93:9.
(Polychaeta, Naidid and Tubificid Oligochaeta, and Hirudinea) of WRONA, F.J., J.M. CULP & R.W. DAVIES. 1982. Macroinvertebrate sub-
North America. Kendall/Hunt Publ. Co., Dubuque, Iowa. sampling: a simplified apparatus and approach. Can. J. Fish. Aquat.
9. KATHMAN, R.D. & R.O. BRINKHURST. 1998. Guide to the Freshwater Sci. 39:1051.
Oligochaetes of North America. Aquatic Resources Center, College WILLIAMS, A.B. 1984. Shrimp, lobsters, and crabs of the Atlantic Coast
Grove, Tenn. of the Eastern United States, Maine to Florida. Smithsonian Insti-
10. MASON, W.T., JR. & P.P. YEVICH. 1967. The use of phloxine B and tution Press, Washington, D.C.
rose bengal stains to facilitate sorting benthic samples. Trans. Amer. BRINKHURST, R.O. 1986. Guide to the Freshwater Aquatic Microdrile
Microsc. Soc. 86:221. Oligochaetes of North America. Canadian Spec. Publ. Fisheries &
4. Bibliography Aquatic Science 84, Dep. Fisheries & Oceans, Ottawa, Ont.
PENNAK, R.W. 1989. Fresh-Water Invertebrates of the United States.
PETTIBONE, M.H. 1963. Marine polychaete worms of the New England Protozoa to Mollusca, 3rd ed. John Wiley & Sons, Inc., New York,
region. I. Families Aphroditidae through Trochochaetidae. U.S. N.Y.
Nat. Mus. Bull. 227:1. VECCHIONE, M., C.F.E. ROPER & M.J. SWEENEY. 1989. Marine Flora and
SMITH, R.I., ed. 1964. Keys to marine invertebrates of the Woods Hole Fauna of the Eastern United States. Mollusca: Cephalopoda. Na-
Region. Contrib. No. 11, Systematics-Ecology Program, Marine tional Marine Fisheries Serv., National Systematics Lab., Washing-
Biological Lab., Woods Hole, Mass. ton D.C.
MCCAIN, J.C. 1968. The Caprellidae (Crustacea: Amphipoda) of the KLEMM, D.J., P.A. LEWIS, F. FULK & J.M. LAZORCHAK. 1990. Macroin-
Western North Atlantic. Smithsonian Institute Bull. 278, Washing- vertebrate Field and Laboratory Methods for Evaluating the Bio-
ton, D.C. logical Integrity of Surface Waters. EPA-600/4-90-030. Environ-
SCHULTZ, G.A. 1969. How to Know the Marine Isopod Crustaceans. mental Monitoring Systems Lab., U.S. Environmental Protection
Wm. C. Brown Company Publ., Dubuque, Iowa. Agency, Cincinnati, Ohio.
HOLME, N.A. & A.D. MCINTYRE. 1971. Methods for the Study of Marine PACKARSKY, B.L., P.R. FRAISSINET, M.A. PENTON & D.J. CONKLIN, JR.
Benthos. IBP Handbook No. 16. Blackwell Scientific Publications, 1990. Freshwater Macroinvertebrates of Northeastern North Amer-
Oxford, England. ica. Cornell University Press, Ithaca, N.Y.
FOSTER, N.M. 1971. Spionidae (Polychaete) of the Gulf of Mexico and KLEMM, D.J. 1991. Taxonomy and pollution ecology of the Great Lakes
the Caribbean Sea. Stud. Fauna Curacao Other Caribbean Islands 36. Region leeches (Annelida: Hirudinea). Mich. Acad. 24:37.
GOSNER, K.L. 1971. Guide to Identification of Marine and Estuarine THORP, J.H. & A.P. COVICH, eds. 1991. Ecology and Classification of
Invertebrates. Cape Hatteras to the Bay of Fundy. Wiley-Inter-
North American Freshwater Invertebrates. Academic Press, Inc.,
science, New York, N.Y.
New York, N.Y.
LEWIS, P.A. 1972. References for the Identification of Freshwater Mac-
LOVELL, L. & R.G. VELARDE. 1991. Regional Standardization of Taxon-
roinvertebrates. EPA-R4-F2-006, U.S. Environmental Protection
Agency, Cincinnati, Ohio. omy. In Proc. Symposium, Biological Criteria: Research and Reg-
BOUSFIELD, E.L. 1973. Shallow-Water Gammaridean Amphipoda of ulation. EPA 440/5-91/005. Off. Water, U.S. Environmental Pro-
New England. Cornell University Press, Ithaca, N.Y. tection Agency, Washington, D.C.
DAY, J.H. 1973. New Polychaeta from Beaufort, with a key to all species CUFFNEY, T.F., M.E. GURTZ & M.R. MEADOR. 1993. Guidelines for the
recorded from North Carolina. U.S. Circ. No. 375, National Oce- Processing and Quality Assurance of Benthic Invertebrate Samples
anic Atmospheric Admin., National Marine Fisheries Serv., Wash- Collected as Part of the National Water-Quality Assessment Pro-
ington, D.C. gram. Open-File Rep. 93-407, U.S. Geological Surv., Raleigh, N.C.
WATLING, L. & D. MAURER. 1973. Guide to the Macroscopic Estuarine MERRITT, R.W. & K.W. CUMMINS, eds. 1996. An Introduction to the
and Marine Invertebrates of the Delaware Bay Region. Delaware Aquatic Insects of North America, 3rd ed. Kendall/Hunt Publishing
Bay Rep. Ser. Vol. 5, p. 178. Univ. Delaware, Newark. Co., Dubuque, Iowa.
10-76 BIOLOGICAL EXAMINATION (10000)

BARBOUR, M.T. & J. GERRITSEN. 1996. Subsampling of benthic samples: BOWMAN, M.F. & R.C. BAILEY. 1997. Does taxonomic resolution affect
A defense of the fixed count method. J.N. Amer. Benthol. Soc. 15: the multivariate description of the structure of freshwater benthic
386. macroinvertebrate communities? Can. J. Fish. Aquat. Sci. 54:1802.
DOBERSTEIN, C.P., J.R. KARR & L.L. CONQUEST. 2000. The effect of
fixed-count subsampling on macroinvertebrae biomonitoring in
small streams. Freshwater Biol. 44:355.

10500 D. Data Evaluation, Presentation, and Conclusions

There are two basic approaches used in evaluating effects of square, Student’s t, regression, correlation, analyses of variance,
pollution on aquatic life. The first is to make a qualitative or nonparametric equivalents).1,2
inventory of the benthic fauna, “above (upstream) and below Mathematical expressions, including numerical indices of
(downstream)” or “before and after” the suspected or known community structure, are useful in characterizing and describing
areas of pollution, thereby determining species presence or ab- aquatic communities. These expressions usually are based on the
sence. Then, through an understanding of the responses of var- structural and functional stability of the system.2
ious species to certain pollutants and habitat degradation, deter- Diversity indices, although limited, condense considerable
mine the significance of damage or change. The second approach biological data into single numerical values.2–9 Unfortunately,
is to make a quantitative analysis of the numbers of individuals, useful information may be lost by condensing biological data.3,10
species, and structure (abundance and composition) of the Select methods for analyzing multivariate benthic community
aquatic community affected by pollution and then to compare data using two important criteria: the methods should test spe-
with reference information. In most pollution surveys these cific impact-related hypotheses suggested by the data quality
approaches are integrated because each provides valuable inter- objectives and study design, and the methods should objectively
pretative information. identify relationships among variables. Use methods that make a
priori assumptions about relationships among variables only
1. Qualitative Data Evaluation
secondarily for presentations, not for primary analysis.
More powerful multivariate statistical analyses generally are
less subject to criticism and may be more appropriate for some
No two aquatic organisms react identically to a pollutant
bioassessment studies.1,10 Recommended data analyses ap-
because of complex relationships between genetic factors and
proaches are: regression of species (or taxa) richness on abun-
environmental conditions. However, certain taxa are relatively
dance, analysis of variance followed by linear orthogonal con-
sensitive to certain types of pollution such as siltation and
trasts,11 various other multivariate approaches (e.g., cluster tech-
turbidity, organic enrichment, acidity, heavy metals and other
niques and ordination, analyzing principal components,
industrial toxic wastes, oil production, agricultural products,
ANOVA, discriminate analyses), and macroinvertebrate com-
radioactive wastes, and thermal effects. For example, operculate
munity metrics2,12 for assessing biomonitoring data and water
snails, immature stages of certain mayflies, stoneflies, caddis-
quality.
flies, riffle beetles, hellgrammites, many marine amphipods,
For statistical evaluation of the data collected in pollution
mysids, bivalve larvae, and echinoderms are sensitive to many
surveys, it always is beneficial to identify the sources of vari-
pollutants. Pollution-tolerant macroinvertebrates such as certain
ability commonly found. Variability in macroinvertebrate data
sludge worms, midge larvae, leeches, pulmonate snails, and some
comes from the methods of sampling and the distribution of
polychaetes usually increase in number under organically enriched
organisms. Perhaps the major source is sampling error. Organ-
conditions. Facultative organisms, those that tolerate moderate pol-
isms generally are clustered in relation to habitat distribution;
lution, include most snails, sowbugs, scuds, and blackfly larvae.
therefore, random samples often show high variability among
Tolerant organisms may be found in either clean or polluted situa-
replicates. In statistical analyses of quantitative data, large num-
tions; thus their presence is not definitive. However, a population of
bers of samples often are required to detect statistically signifi-
tolerant organisms combined with an absence of intolerant ones is a
cant differences. Exercise care in using parametric statistical
good indication of the presence of pollution. The same species
methods because the basic assumption of normal distribution is
found in different geographical areas may well react differently or
not always true. Data often have to be transformed before being
be present in different numbers throughout the year.
tested. Do not assume that a statistically significant difference is
ecologically significant.
2. Quantitative Data Evaluation

Statistical methods of data evaluation and mathematical de- 3. Data Presentation

scription of community structure are valuable tools in data
analysis. Analysis of biological data commonly begins with the Data presentation may take many forms. The basic techniques
calculation of descriptive statistics (mean, standard deviation, include tables, bar graphs (horizontal and vertical), pie diagrams,
standard error, and confidence intervals). Analysis proceeds by pictorial charts (ideographs), line graphs, frequency distribution
application of robust statistical methods of comparison (Chi- tables and graphs, histograms, frequency polygons, and cumu-
BENTHIC MACROINVERTEBRATES (10500)/Data Evaluation, Presentation, and Conclusions 10-77

lative frequency polygons. These may be superimposed on maps. INGRAM, W.M. 1960. Effective methods for collecting and recording data
Several reports that may be useful in analyzing macroinverte- from water pollution surveys. In C.M. Tarzwell, compiler. Biolog-
brate data have been included in the bibliography. Methods for ical Problems in Water Pollution, p. 260. U.S. Dep. Health, Edu-
interpreting benthic invertebrate data with measures of contam- cation & Welfare, Cincinnati, Ohio.
ination and toxicity are available.13 INGRAM, W.M. & A.F. BARTSCH. 1960. Graphic expression of biological data
in water pollution reports. J. Water Pollut. Control Fed. 32:297.
4. Conclusions PIELOU, E.C. 1966. The measurement of diversity in different types of
biological collections. J. Theor. Biol. 13:131.
Despite detailed data quality objectives, field methodology, CAIRNS, J., JR. 1971. A simple method for the biological assessment of
the effects of waste discharges on aquatic bottom-dwelling organ-
and laboratory analysis and data presentation, it often requires
isms. J. Water Pollut. Control Fed. 43:755.
extensive professional experience and skill and knowledge of the
ERMAN, D.C. & W.T. HELM. 1971. Comparison of some species impor-
scientific literature to draw defensible conclusions from a data tance values and ordination techniques used to analyze benthic
set. Even in the best circumstances, there can be more than one invertebrate communities. Oikos 22:240.
conclusion drawn from a study. When more than one conclusion ORLOCI, L., C.R. RAO & W.M. STITELER, eds. 1978. Multivariate Meth-
is possible, it is appropriate to present all options. ods in Ecological Work. International Cooperative Publ. House,
Fairland, Md.
5. References CONOVER, W.J. 1980. Practical Nonparametric Statistics, 2nd ed. John
Wiley & Sons, New York, N.Y.
1. GREEN, R.H. 1979. Sampling Design and Statistical Methods for POLLARD, J.E. 1981. Investigator differences associated with a kicking
Environmental Biologists. John Wiley & Sons, New York, N.Y. method for sampling macroinvertebrates. J. Freshwater Ecol. 1:215.
2. KLEMM, D.J., P.A. LEWIS, F. FULK & J.M. LAZORCHAK. 1990. Mac- GAUCH, H.G., JR. 1982. Multivariate Analysis in Community Ecology.
roinvertebrate Field and Laboratory Methods for Evaluating the Cambridge University Press, New York, N.Y.
Biological Integrity of Surface Waters. EPA-600/4-90-030. Envi- PLATTS, W.S., W.F. MEGAHAN & G.W. MINSHALL. 1983. Methods for Eval-
ronmental Monitoring Systems Lab., U.S. Environmental Protection uating Stream, Riparian, and Biotic Conditions. General Technical
Agency, Cincinnati, Ohio. Rep. INT-138. U.S. Dept. Agriculture, U.S. Forest Serv., Ogden, Utah.
3. WASHINGTON, H.G. 1984. Diversity, biotic and similarity indices: a ALLAN, J.D. 1984. Hypothesis testing in ecological studies of aquatic
review with special reference to aquatic ecosystems. Water Res. insects. In V.H. Resh & D.M. Rosenberg, eds. The Ecology of
18:653. Aquatic Insects, p. 484. Praeger Scientific, New York, N.Y.
4. WILHM, J.L. 1967. Comparison of some diversity indices applied to ZAR, J.H. 1984. Biostatistical Analysis, 2nd ed. Prentice-Hall, Inc.,
populations of benthic macroinvertebrates in a stream receiving Englewood Cliffs, N.J.
organic wastes. J. Water Pollut. Control Fed. 39:1673.
FAUSCH, D.D., J.R. KARR & P.R. YANT. 1984. Regional application of an
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6. WILHM, J.L. 1970. Range of diversity index in benthic macroinver-
DAWSON, C.L. & R.A. HELLENTHAL. 1986. A Computerized System for
tebrate populations. J. Water Pollut. Control Fed. 42:R221.
the Evaluation of Aquatic Habitats Based on Environmental Re-
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quirements and Pollution Tolerance Association of Resident Organ-
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Agency, Cincinnati, Ohio.
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