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BIOMONITORING PROGRAM

Massachusetts Department of Environmental Quality Engineering

DIVISION of WATER POLLUTION CONTROL

Thomas C. McMahon, Director

BIOMONITORING PROGRAM

STANDARD OPERATING PROCEDURES

1987

Technical Services Branch

Massachusetts Division of Water Pollution Control

Department of Environmental Quality Engineering

Westborough

Executive Office of Environmental Affairs
James S. Hoyte, Secretary

Department of Environmental Quality Engineering
S. Russell Sylva, Commissioner

Division of Water Pollution Control
Thomas C. McMahon, Director

April 1987

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^J^^^^^^^^^^^^^^^^^^r

4.0 BIOMONITORING PROGRAM

SECTION PAGE

1.0 INTRODUCTION AND PURPOSE 1

2.0 BIOMONITORING SURVEY PROGRAM ELEMENTS 3

2.1 Stream Classification 4

2.2 Aquatic Macroinvertebrate Rapid Bioassessment 11

2.3 Site Assessment 16

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS 21

3.1 Phytoplankton 22

3.2 Periphyton 36

3.3 Aquatic and Wetland Vegetation 45

3.4 "Aquatic Macroinvertebrates 54

3.5 Fish 67

3.6 Microtox™ Analysis 81

3.7 Chlorophyll Analysis 89

4.0 QUALITY ASSURANCE 96

5.0 GENERAL BIOLOGICAL FIELD AND LABORATORY REFERENCES 101

in

1.0 INTRODUCTION AND PURPOSE

1.0 INTRODUCTION AND PURPOSE

It is the goal of the Federal Clean Water Act (PL 95-217) to restore and
maintain the biological integrity of the nation's waters. Biological
monitoring provides the most reliable measure of the attainment of this
goal, i.e., water quality that provides for the protection and propagation
of fish, shellfish and wildlife.

Sampling and analyzing aquatic life provides information on water quality
that can easily escape standard physico-chemical sampling. The organisms
themselves are efficient in-stream monitors, for their lives reflect the
cumulative impact of pollution on the waterbody. They are valuable in
revealing transient pollution episodes such as oil spills and brief
dissolved oxygen sags. For the same reason they are the best means of
measuring long term trends in a waterbody. In addition, the presence of
specific indicator organisms may infer the presence of particular chemicals
not included in routine analysis or in quantities below detection limits
of chemical testing.

Aquatic biota are usually collected and analyzed by community. These
communities include plankton, periphyton, raacrophyton, macroinvertebrates
and fish. The communities are used alone or in combinations to assess
specific water quality problems such as thermal pollution, toxics, and
eutrophication. The analysis of the samples includes taxonomic
identification for diversity indices, water quality indices, trophic level
and indicator organism analysis. Plant pigments are extracted for
chlorophyll analysis and animal tissues are tested for bioconcentration of
chemicals. The overall health and appearance of the organisms is used to
detect chronic toxicity and genotoxic effects (carcinogens, mutagens and
tetratogens) . Standard laboratory organisms are also used in situ and in
vitro to measure toxicity. Bacteria, algae, macroinvertebrates and fish
are all commonly used for this purpose.

Biological monitoring can be more cost effective than chemical screening,
more reliable at measuring total pollutant loads, more sensitive to extreme
conditions and more faithful to the goal of the Act, than other forms of
monitoring. However, the relationship between the biota and the
environment is subtle and complex and by no means completely understood.
Results of biological investigations are often qualitative, and even
quantitative studies are open to interpretation. Therefore biological
monitoring data are used to complement physico-chemical data and not
replace them.

The methods of monitoring and analysis are evolving and may differ among
investigators. At best, procedures used by the Division of Water Pollution
Control are fully documented in this Standard Operating Procedures docu-
ment, so that those attempting interpretation will be fully informed, and
temper their conclusions accordingly.

2.0 BIOMONITORING SURVEY PROGRAM ELEMENTS

SECTION PAGE

2.1 STREAM CLASSIFICATION 5

2.1.1 Introduction and Purpose 5

2.1.2 Objectives 5

2.1.3 Approach 5

2.1.4 Parametric Coverage 5

2.1.5 Data Record Sheets 6

2.1.6 References 10

STREAM CLASSIFICATION

2.1 STREAM CLASSIFICATION

2.1.1 INTRODUCTION AND PURPOSE

This program has been developed to systematically sample and classify
the Commonwealth's rivers and streams. Each survey qualitatively
provides documentation of a specific watercourse's physical and chemical
characteristics and predominant biological components. These data can be
used - on a stream or site-specific basis - to determine water-use
classifications in accordance with Massachusetts Surface Water Quality
Standards.

2.1.2 OBJECTIVES

1. To identify, demonstrate, and standardize methods and procedures
for the collection and analyses of stream habitat data;

2. to characterize rivers, streams, and related aquatic habitats
(e.g., river impoundments) hydrophysically and chemically;

3. to qualitatively document the dominant floral and faunal
components - or communities - of streams and stream-side habitats;

4. to segment and classify rivers and streams into major habitats for
the purpose of water-use designation;

5. to provide supplementary information to other programs to aid in
regulatory and enforcement actions, and evaluating special problems;
and

6. to collect and reference plant and animal specimens for future
study, and determine their state-wide distribution.

2.1.3 APPROACH

Preliminary planning and analysis first divides the river or stream into
longitudinal zones - or subsystems, i.e., tidal, lower perennial, upper
perennial, intermittent, and others (e.g., canals, ditches) - according
to morphometric and hydrologic characteristics derived from USGS
topographic maps. Physico-chemical and biological field collections are
made, in most instances, at locations - or sites - determined after
initial evaluation and field reconnaissance (see: "Data Record Sheets").
Specific sampling locations are arranged to cover significant and
representative lotic-water and other related macrohabitats . Field dates,
particularly for biological sampling, are generally during the period
April to October, in order to take advantage of plant and animal
availability. All field sampling is qualitative in nature, unless
special needs dictate otherwise. Data collected are recorded for each
community on individual standard field sheets (see: "Biological Field
and Laboratory Methods").

2.1.4 PARAMETRIC COVERAGE

Physical and chemical data are collected, including: stream reach width
and depth; stream reach and floodplain substrate character; stream
temperature; water transparency; and water chemistry. Sampling of
phytoplankton and periphyton, aquatic vascular plants, streamside and
riparian vegetation, and aquatic macroinvertebrates is performed at each

2.1.5 DATA RECORD SHEETS

STREAM CLASSIFICATION

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2.1.6 REFERENCES

1. Cowardin, L.M., V. Carter, F.C. Golet, and E.T. LaRoe. 1979. Classifi-
cation of Wetlands and Deepwater Habitats of the United States. FWS/OBS
79/31. Office of Biological Services, United States Fish and Wildlife
Service, Washington, D.C. vi + 103 p.

2. Marmelstein, A. 1978. Classification, Inventory, and Analysis of Fish
and Wildlife Habitat: Proceedings of a National Symposium held at
Phoenix, Arizona, 24-27 January 1977. FWS/OBS-78/76. Office of
Biological Services, U.S. Fish and Wildlife Service, Washington, D.C.
vi + 604 p.

3. Platts, W.S., W.F. Megahan, and G.W. Minshall. 1983. Methods for
Evaluating Stream, Riparian, and Biotic Conditions. General Technical
Report INT-138. Intermountain Forest and Range Experiment Station,
United States Forest Service, Ogden, Utah. ii + 70 p.

4. Sather, J.H., (ed). 1976. Proceedings of the National Wetland
Classification and Inventory Workshop held at the University of Maryland,
College Park, Maryland, 20-23 July 1975. FWS/OBS-76/09 . Office of
Biological Services, U.S. Fish and Wildlife Service, Washington, D.C.

vi + 110 p.

5. Terrell, T.T. and W.J. McConnell, (eds). 1978. Stream Classification -
1977: Proceedings of a Workshop held at Pingree Park, Colorado, 10-13
October 1977. .FWS/OBS-78/23 . Biological Services Program, U.S. Fish and
Wildlife Service, Fort Collins, Colorado. iv + 45 p.

10

MACRO INVERTEBRATE RAPID BIOASSESSMENT

SECTION PAGE

2.2 AQUATIC MACRO INVERTEBRATE RAPID BIOASSESSMENT 12

2.2.1 Introduction and Purpose 12

2.2.2 Objectives 12

2.2.3 Approach 12

2.2.4 Parametric Coverage 14

2.2.5 References 15

11

2.2 AQUATIC MACRO INVERTEBRATE RAPID BIOASSESSMENT

2.2.1 INTRODUCTION AND PURPOSE

Macroinvertebrate rapid bioassessment (MRB) surveys involve the use of
qualitative and semiquantitative sampling methods designed to minimize
laboratory time requirements for taxonomic identification and enumera-
tion of aquatic macroinvertebrate organisms.

2.2.2 OBJECTIVES

1. To provide standardized methods and procedures for assessing the
impacts of toxic and conventional organic pollution on aquatic
macroinvertebrates ;

2. to obtain reliable biological water quality information to supple-
ment the collection of standard physico-chemical water quality
data; and

3. to provide the basis for making relative comparisons pertaining to
water quality conditions between sampling stations and/or to
document long-term trends at fixed sites.

2.2.3 APPROACH

While rapid bioassessments make use of the qualitative analysis of
periphyton, aquatic and wetland vegetation, and fish communities,
specific semi-quantitative sampling and analytical methods have been
developed for use in assessing the macroinvertebrate community.

An upstream-downstream sampling regime is employed whereby known or
suspected sources of pollution are bracketed by sampling stations.
Selected aquatic communities are assessed and compared with unimpacted
control (or reference) communities. Conclusions relative to water
quality condition are drawn from a knowledge of the environmental
requirements and pollution ecology of the individual taxa or assemblages
encountered.

For macroinvertebrate rapid bioassessment the components of a 100 organism
subset are identified to genus or species level whenever possible. The
taxonomic data are then compiled to determine the status of the various
criteria used to rank water quality. These criteria include:

1. Species richness;

2. distribution "balance";

3. the EPT value;

4. percent contribution, pollution tolerances, and feeding habits
of the five numerically dominant species;

5. Hilsenhoff Biotic Index (HBI).

1-2

MACROINVERTEBRATE RAPID BIOASSESSMENT

Field observations were also considered, as they often reveal important
factors contributing to the quality of the benthic community.

Species richness, the number of different kinds of organisms present,
will tend to decrease in response to pollution while the distribution of
individuals becomes uneven, or unbalanced. That is to say, under the
influence of pollution benthic macroinvertebrate communities become less
diverse, with the majority of individuals falling into fewer taxa
(Tarzwell and Gaufin 1953, Bartsch and Ingram 1959, Weber 1973, Hawkes
1979, and Welch 1980). By examining the relative contribution of the
five numerically dominant taxa the evenness of the distribution can be
judged.

The pollution tolerances of the dominant community members can be
revealing as to the degree of pollution impacting a stream. Likewise,
the number of species present from the orders Ephemeroptera, Plecoptera,
and Trichoptera can be tabulated to formulate the "EPT value." These
orders are composed of species that are regarded as intolerant or facul-
tative in response to enrichment with conventional pollutants — Plecoptera
are all intolerant, Ephemeroptera and Trichoptera have both intolerant
and facultative members (Weber 1973, Hilsenhoff 1982). Also of impor-
tance are the feeding habits of the dominant taxa, as these will reflect
community shifts to exploit the food source available, e.g., a filter
feeding community downstream of an effluent high in suspended solids.

Hilsenhoff (1982) developed an index (HBI) based on the tolerances of
aquatic macroinvertebrates to pollution with conventional organics.
While his sampling protocol was similar to the one used here, he restric-
ted his analysis to aquatic arthropods dependent on dissolved oxygen.
The MRB, on the other hand, makes use of aquatic annelids and mollusks
for the information they may contribute in attempts to evaluate the
impacts of various types of pollution. Consequently, if the HBI is to be
used as part of the MRB it becomes necessary to assign tolerance values
to organisms excluded by Hilsenhoff as well as any regionally unique
aquatic arthropod taxa that otherwise would have been included by
Hilsenhoff. Since Hilsenhoff 's tolerance values range from zero
(intolerant) to five (tolerant) and most literature provides information
on pollution tolerances as tolerant, facultative, and intolerant, assign-
ing new values was difficult. Lacking any better information the
assigned values then became: intolerant=l , facultative=2. 5 , and
tolerant=4. These modifications surely weaken the reliability of the
HBI, if not by using dubious tolerance values, then at least by virtue of
eliminating the sensitivity to the extremes. Nonetheless, with these
considerations in mind the HBI is retained in the MRB because if the
index value falls at one of the extremes it indicates either very little
DO stress (HBI<2) or very serious DO stress (HBI>4).

The MRB guidelines identify the range of characteristics indicative of
different levels of pollution as follows:

1. Non-Impacted - Diverse fauna, at least 30 species in riffle habi-
tats. Biotic index about 2.00. Mayflies, stoneflies, and caddis-
flies are well-represented, EPT value greater than 10. Dominant
species are intolerant or facultative; no species comprises more

13

than 25% of Che individuals; oligochaete worms comprise less than
of the individuals.

2. Slightly Impacted - Species richness usually 20-30. Biotic index
2.00-3.00. Mayflies and stoneflies may be restricted, EPT value
6-10. Dominant species are mostly facultative. Fauna often not so
well balanced, often with one species comprising more than 25% of
the individuals; oligochaete worms may comprise more than 20% of the
individuals .

3. Moderately Impacted - Species richness 10-20. Biotic index 3.00-
4.00. Mayflies and stoneflies rare or absent, caddisflies often
restricted, EPT value 2-5. Dominant species are facultative or
tolerant. Oligochaetes often comprise at least 20% of the
individuals .

4. Severely Impacted - Species richness less than 10. Biotic index
greater than 4.00. Mayflies, stoneflies, and caddisflies rare or
absent, EPT value 0-1. Fauna often restricted to midges and worms.
Dominant species are almost all tolerant. Fauna usually greatly
imbalanced, with dominant species comprising more than 35% of the
individuals .

These are generalizations about complex ecosystems and may not always
result in complete agreement of all parameters. In such cases it is
necessary to select a category based on a consensus of the majority of
indicators. It is also necessary to consider the integrity of each
component so that those possibly influenced by factors other than
pollution can be de-emphasized, or if appropriate, eliminated from the
assessment. For instance, a data set may contain 21 species, no species
representing more than 25% of the community, oligochaetes comprising 21%,
an EPT value of three, an HBI of 3.25, with four of the five dominant
species being facultative, and the fifth being tolerant. Knowing that
the data set includes significant numbers of aquatic annelids and
mollusks, the HBI should not weigh heavily in the analysis. A review of
the other criteria would tend toward a rating of "slightly impacted" for
this hypothetical community.

2.2.4 PARAMETRIC COVERAGE

Rapid assessment surveys include, at a minimum, semi-quantitative aquatic
macroinvertebrate sampling and water temperature determinations. However,
qualitative analyses of the algae, macrophyte, and fish communities may
also be conducted. Often, flow measurements, substrate characterization,
and water chemistry sampling are conducted to supplement the results of
biological sampling.

14

MACRO INVERTEBRATE RAPID BIOASSESSMENT

2.2.5 REFERENCES

1. Bartsch, A.F. and W.M. Ingram. 1959. Stream Life and the Pollution
Environment. Public Works. 90:104-110.

2. Beck, W.M., Jr. 1977. Environmental Requirements and Pollution
Tolerance of Common Freshwater Chironomidae. United States Environmental
Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio. EPA-600/4-77-024. vi + 261 p.

3. Bilger, M.D. 1986. A Preliminary Checklist of the Aquatic Macro-
invertebrates of New England, Including New York State. U.S. Environmental
Protection Agency, Environmental Services Division, Lexington,
Massachusetts. viii + 72 p.

4. Harris, T.L. and T.M. Lawrence. 1978. U.S. Environmental Requirements
and Pollution Tolerance of Trichoptera. U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati.
EPA-600/4-78-063. vi + 310 p.

5. Hart, C.W., Jr. and S.L.H. Fuller, (eds.). 1974. Pollution Ecology of
Freshwater Invertebrates. Academic Press, Inc., New York. xiv + 389 p.

6. Hawkes, H.A. 1979. Invertebrates as Indicators of River Quality. In:
Biological Indicators of Water Quality (A. James and L. Evison, eds.).
John Wiley and Sons, Inc., New York. pp. 2.1-2.45.

7. Hilsenhoff, W.L. 1982. Using a Biotic Index to Evaluate Water Quality
in Streams. Technical Bulletin No. 132. Wisconsin Department of Natural
Resources, Madison. 22 p.

8. Tarzwell, CM. and A.R. Gaufin. 1953. Some Important Biological Effects
of Pollution Often Disregarded in Stream Surveys. Proc. 8th Industrial
Waste Conference, Purdue University Engineering Bulletin. pp. 295-316.

9. Weber, C.I.., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. U.S. Environmental
Protection Agency, National Environmental Research Center, Cincinnati.
EPA-670/4-73-001. xii + 146 p. + appendices.

10. Welch, E.B. 1980. Ecological Effects of Waste Water. Cambridge
University Press, New York. xii + 337 p.

15

SECTION PAGE

2.3 SITE ASSESSMENT 17

2.3.1 Introduction and Purpose 17

2.3.2 Objectives 17

2.3.3 Approach 17

2.3.4 Parametric Coverage 18

2.3.5 Quantitative Data Analyses 18

2.3.6 References 20

16

SITE ASSESSMENT

2.3 SITE ASSESSMENT

2.3.1 INTRODUCTION AND PURPOSE

While site assessments make use of a number of qualitative and semi-
quantitative methods borrowed from stream classification and/or rapid
assessment protocols, they may also be expanded to include quantitative
sampling and analytical procedures. In fact, site assessment surveys
may range in scope from a qualitative assessment of the impact of a
single wastewater discharge on a single aquatic community to intensive
quantitative assessments of one or more communities. The latter are
labor and resource intensive and are limited to those situations where
the need exists for statistically derived statements of confidence in
the results.

2.3.2 OBJECTIVES

1. To provide an adequate data base for making quantitative determina-
tions of standing crop, biomass, or measures of community structure
such as species diversity and richness;

2. to provide sufficient data for testing for significant differences
between communities using appropriate statistical methods;

3. to provide standard methods for assessing the impacts of pollution
on aquatic biota and water uses; and

4. to supplement physico-chemical water quality data with biological
information.

2.3.3 APPROACH

Whenever possible, sampling stations are located upstream and downstream
from known or suspected sources of pollution or other factors that
might impact water quality conditions. The underlying assumption is made
that, if all other environmental factors remain constant, a change in
water chemistry will alter downstream community structure or biomass.
Therefore, impact assessment is carried out by making community
structural comparisons between upstream or nearby reference communities
and downstream communities.

Measures of community structure to be employed are selected on a case-by-
case basis according to the requirements of individual site assessments.
Parameters include 1) abundance; 2) taxonoraic richness; 3) evenness; and
4) diversity (e.g., Shannon Weaver H1). Comparisons of communities
between sites are made using the above measures and standard significance
tests such as t-tests.

Less intensive site assessments involving the use of qualitative or semi-
quantitative techniques are conducted according to the methods presented
in previous sections for stream classification and rapid assessment
surveys.

17

2.3.4 PARAMETRIC COVERAGE

Site assessments may involve the use of qualitative, semi-quantitative,
or quantitative analyses of one or more of the following communities:
phytoplankton; periphyton; macrophyton; macroinvertebrates ; or fish.
Biological stream sampling is supplemented, as deemed appropriate, by
hydrological and physico-chemical assessments such as the determination
of stream width, depth, flow, water temperature, substrate charac-
terization, and chemical analyses.

2.3.5 QUANTITATIVE DATA ANALYSES

Definitions of some of the more commonly used indices of community
structure are presented below.

Abundance

Two abundance measures are often used: (1) the sum total of individuals
found in all taxonomic groups in a particular data set (termed "total
numbers"); and (2) the relative proportion of individuals found in
different taxonomic categories (termed "relative abundance").

If a relationship between productivity and numbers of individuals can
be established, increases from control to test sites in the total number
of organisms found may be a result of increased nutrient availability.
Decreases in this measure may be related to changes in nutrients and/or
the influence of toxic substances. Changes in the relative abundance of
major taxonomic groups may be related to habitat alterations between
sites. When changes in the relative abundance of major groups are
accompanied by a decrease in richness (see below) they may be due to
either changes in nutrient availability and/or to toxic stress.

Taxonomic Richness

This term refers to the number of different taxonomic groups in a par-
ticular sample. Comparisons of richness are based on the assumption that
physiological stress (defined as those instances under which
environmental conditions such as temperature, oxygen concentration, pH,
etc., exceed the tolerance limits of an individual) due to a toxic
discharge can reduce the number of taxa originally inhabiting a certain
area.

Richness of a sample collection is positively correlated with sampling
effort. As area sampled, time spent sampling, and/or number of organisms
collected are increased, the number of different taxa encountered also
increases. For these reasons, comparisons should only be made between
data sets for which sampling efforts are similar or nearly so.

Evenness

This is a measure of the distribution of individual organisms over
different taxonomic categories. Most evenness indices range from a value
of zero to 1.0, with a completely uniform distribution yielding a value
of 1.0.

18

SITE ASSESSMENT

Diversity indices (see below) compress richness and evenness into a
single number. However, information is lost in this process. In an
attempt to regain some of this information, ecologists have used evenness
or equitability ratios that are usually of the form: measured diversity/
standard diversity, where the latter term is the maximum diversity of
a community given a certain richness value. A basic problem with this
approach is that the value or the ratio is dependent upon the particular
characteristics of the diversity index. Thus, biases inherent to the
index are incorporated into, and perhaps magnified by, the evenness
ratio.

Diversity Indices

Most diversity indices attempt to interdigitate and refine two components
of community structure: richness and evenness.

The Shannon Weaver H* is commonly used for two reasons: (1) it is simple
in form; and (2) it has a known variance structure. Due to the latter
attribute, a t-test for differences in R^ between two data sets can be
run. The form of the index and its variance structure are taken from
Poole (1974) and are presented below.

H =

- ^ pi. In pi

S-l
"ZF"

where

Var. H' =

- s

Pi

.m2

pi -

r

s

>» pi.

In

>
Pi

i=l

i=l

)

N

S-l

2
21T

S
Pi

N =

number of taxa

the proportion of the

total number of

individuals consisting

of the i1-" taxon

total number of

individuals

Another diversity index commonly used is Simpson's Index which can be
defined as: D = 1 - C

where C = f"

i-1

ni(ni-l)
N(N-l)

and S =
ni =

N =

as above

the number of

individuals in the

i*-" species

as above

The term C is an approximation of the probability that two individuals
drawn at random from a population of N individuals will belong to the
same taxon. The higher this probability, the lower the "diversity"
(as measured by this index) of the collection; hence D (equal to 1-C)
is used as the index since this parameter will increase with the
"diversity" of the sample.

The two indices cited above differ in their sensitivity to changes in
richness and evenness. Whereas the Shannon Weaver Index is more an
expression of the overall evenness of the community, the Simpson's Index
expresses the relative degree of dominance of a few taxa in the
community.

19

2.3.6 REFERENCES

1. Bilger, M.D. 1986. A. Preliminary Checklist of the Aquatic Macro-
invertebrates of New England, Including New York State. U.S. Environmental
Protection Agency, Environmental Services Division, Lexington,
Massachusetts, vii + 72 p.

2. Edmondson, W.T. and G.G. Winberg, (eds.). 1971. A Manual of Methods for
the Assessment of Secondary Productivity in Freshwaters. IBP Handbook
No. 17. Blackwell Scientific Publications, Oxford, England, xxiv +

358 p.

3. Hart, C.W., Jr. and S.L.H. Fuller, (eds.). 1974. Pollution Ecology of
Freshwater Invertebrates. Academic Press, New York. xvi + 389 p.

4. Hynes, H.B.N. 1974. The Biology of Polluted Waters. University of
Toronto Press, Ontario, Canada, xiv + 202 p.

5. MacKenthun, K.M. 1969. The Practice of Water Pollution Biology. United
States Department of the Interior, Federal Water Pollution Control
Administration, Washington, D.C. xii + 281 p.

6. Poole, R.W. 1974. An Introduction to Quantitative Ecology.
McGraw-Hill, Inc., New York. xii + 532 p.

7. . Simpson, E.H. 1949. Measurement of Diversity. Nature 163: 688.

8. Warren, C.E. 1971. Biology and Water Pollution Control. W.B. Saunders
Company, Philadelphia. xvi + 434 p.

9. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA-670/4-73-001 ,
United States Environmental Protection Agency, National Environmental
Research Center, Cincinnati, Ohio. xii + 146 p. + appendices.

10. Welch, E.B. 1980. Ecological Effects of Wastewater. Cambridge
University Press, England. xii + 337 p.

20

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

21

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3 . 1 Phytoplankton 23

3.1.1 Definition 23

3.1.2 Objectives 23

3.1.3 Field Sampling 23

3.1.4 Laboratory Analysis 24
Sample Preservation 24
Phytoplankton Examination 25

3.1.5 Field Equipment and Supply List 30

3.1.6 Data Record Sheets 31

3.1.7 References 34

22

PHYTOPLANKTON
3 . 1 PHYTOPLANKTON

3.1.1 DEFINITION: Phytoplankton are the algae of lakes and large rivers that
live suspended in the water. They are chlorophyll-bearing, unicellular
organisms which have no true roots, stems, or leaves. They occur in
free-living, colonial, frond-like or filamentous forms and vary in size
from unicells 0.5 microns in diameter to the macroscopic seaweeds. Algae
are generally grouped into the Divisions (and classes) Euglenophyta
(Euglenophyceae) ; Chlorophyta (Chlorophyceae, Charophyceae) ; Rhodophyta
(Rhodophyceae) ; Cyanophyta (Myxophyceae) ; Pyrrophyta (Desmokantae ,
Dinophyceae) ; Chrysophyta (Xanthophyceae, Chrysophyceae ,
Bacillariophyceae) ; Phaeophyta (Phaeophyceae) ; and Cryptophyta
(Cryptophyceae) .

3.1.2 OBJECTIVES

1. To document the existing phytoplankton community and determine
long-term (yearly) and short-term (seasonal) trends;

2. to evaluate direct effects on water composition including dissolved
oxygen, pH, hardness, and optical properties;

3. to assess conditions affecting the general condition of water
quality including noxious and toxic conditions, offensive tastes
and odors ;

4. to identify indicators of trophic status, organic enrichment and
specific chemical contamination; and

5. to quantify autotrophic bioraass and make inferences concerning
productivity.

3.1.3 FIELD SAMPLING

Samples for phytoplankton analyses are collected in clean one-liter
bottles made of plastic or glass, that have been rinsed with sample water.
Approximately one-half liter of sample water is collected.

In rivers that are mixed vertically and horizontally, samples are
collected midstream 0.5 to 1.0 meters (m) below the surface. In lakes
and impoundments, samples are collected at the "deep-hole" station. If
the lake is thermally unstratified the sample is collected 0.5-1.0 m
below the surface. If the lake is thermally stratified, an integrated
column sample is collected by lowering a one centimeter (approximately)
ID plastic tube (with a weight attached) to the thermocline zone, pinched
below the miniscus and raised into the boat. The sample is then drained
into a clean and rinsed collection bottle. This procedure is repeated
until one-half liter of water is collected. All samples are cooled to
4°C and placed in the dark following collection.

For special studies in riverine and lacustrine habitats, samples are
collected from major depth zones or water masses. Sampling depths at
each site are determined by specific conditions. In shallow areas (2-3
m) , subsurface sampling is generally conducted. In deeper areas
samples are collected at regular intervals at depths throughout the
euphotic zone.

23

Pertinent information collected and recorded in the field includes
meteorological data (cloud cover, wind speed and direction, air tempera-
ture); surface water conditions; water color, turbidity, odors; total
depth at station; and other descriptive information.

The frequency of sampling is dependent on the intent of the study as well
as the range of seasonal fluctuations, the immediate meteorological
conditions, adequacy of equipment, and availability of personnel. In
tidally-inf luenced habitats, phytoplankton samples are collected at all
tide stages, particularly at the end and the beginning of both the flood
and ebb tides.

3.1.4 LABORATORY ANALYSES

Sample Preservation

Phytoplankton samples collected in the field are cooled to 4°C and kept
in the dark in transit to the laboratory. Upon arrival at the
laboratory, they are placed in a refrigerator until further processing.
Samples are generally analyzed on the day of collection. Samples not
analyzed on the day of collection are stored in a refrigerator overnight
with the caps loosened to allow gas exchange. Samples stored for more
than 48 hours are fixed by the following methods and preservatives:

1. Lugol's solution: For short-terra storage, 0.3 ml Lugol's solution
is added per 100 ml of sample aliquot and. stored in the dark. For
long-term storage, 0.7 ml Lugol's solution is added per 100 ml of
sample. [Lugol's solution is prepared by dissolving 20 grams (g)
potassium iodide (KI) and 10 g iodine crystals in 200 ml distilled
water containing 20 ml glacial acetic acid],

2. Formalin: To preserve samples, 40 ml buffered formalin is added
to one liter of sample.

3. M-* Fixative: For preservation, 20 ml M-* fixative is added to
one liter of sample and stored in the dark. [M^ is prepared by
dissolving 5 g KI , 10 g iodine, 50 ml glacial acetic acid, and 250
ml formalin in one liter of distilled water].

Color -

Cupric sulfate solution is added to the sample to preserve color [Cupric
solution is prepared by dissolving 21 g cupric sulfate in 100 ml
distilled water] .

Clumping -

To prevent clumping, a detergent solution is added to the sample [20 ml
liquid detergent is added to 100 ml distilled water].

24

PHYTOPLANKTON

Phytoplankton Examination

Log-In Procedure -

1) Each sample is assigned a number and logged in as it is brought into
the laboratory. The numbers are in consecutive order and are recorded
both on the sample tag and in a notebook (log book).

2) Next to the number in the log book are also recorded the station
number and location, date collected, date analyzed, initials of
collector, type of samples, sample depth, and analyses requested,
i.e., chlorophyll and/or algal identifications.

Phytoplankton Examination Equipment List -

1) Microscope - capable of 200x power with working distance greater than
1 mm.

2) Sedgwick-Raf ter (S-R) counting cells

3) Whipple micrometer reticule

4) Stage micrometer

5) Pipettes

6) Bench sheets

7) Lens paper

Procedure for Filling the Sedgwick-Raf ter Cell:

1) Place the cover glass diagonally across the cell.

2) Use large-bore 1 ml pipette to fill the S-R cell.

3) Place tip of the pipette in the corner of the S-R cell and slowly
release the pressure of your finger on the end of the pipette. The
cover slip will then rotate and cover the sample.

4) To reduce error:

a. Do not overfill the cell which would yield a depth greater than
1 mm.

b. Do not allow large air bubbles to form. To prevent the formation
of these air spaces, a drop of distilled water is placed on the
edge of the cover glass occasionally during the microscopic
examination.

25

Procedure for Phytoplankton Examination:

1) Shake the sample bottle to mix well.

2) Rinse 1 ml pipette with distilled water (inside and out) and three
times with sample water.

3) Fill counting cell with 1 ml of sample water (see: "Procedure for
Filling the Sedgwick-Raf ter Cell").

4) Allow sample to settle for 15 minutes (the settling rate for algae is
4 mm/hr; since the depth of the counting cell is 1 mm, a 15 minute
settling time is used.

5) While sample is settling, prepare a microscopic slide or Palmer cell
which will allow you to view the sample at a higher power. List the
algal genera identified.

6) Use the keys to determine unknown organisms; particularly dominant
ones.

7) Scan the Sedgwick-Raf ter counting cell at 4x and determine need for
concentration or dilution.

8) At 200x find the edge of the counting cell and focus on the top of
the cell. Continue turning the coarse focusing knob on the microscope
until the bottom of the cell comes into focus.

9) At least two strips in the S-R counting cell must be counted.

10) Counts are done on both the bottom of the cell and the top or
underside of the cover slip.

11) Identify and count all the algae that are located in the Whipple grid.
Algae which are half in and half out of the top of the grid should be
included in the count. Algae which are half in and half out of the
bottom of the grid are not included in the count.

12) If the algal density appears to be high then fields can be counted
instead of strips. A field is represented by a Whipple grid. Ten
fields on two slides are counted and then averaged.

13) A strip is represented by the width of Whipple grid and the length of
a Sedgwick-Raf ter cell.

Explanation of the Phytoplankton Examination Sheet:

(Refer to: "Phytoplankton Examination" Form)

1) Line 1 - station location, station number, date of collection

2) Line 2 - initials of analyst, milliliters of sample, which will be
either 1 ml or the total concentrated, type of count, i.e., fields or
strips and the date of analysis.

26

PHYTOPLANKTON

3) Lab number - the number assigned the sample by the investigating
laboratory (see: "Log-In Procedures").

4) Bottom two lines - chlorophyll in mg/m^, total live algae
(cells/ml), multiplication factor (S-R) for the particular microscope
and power used, microscope manufacturer and type, the microscope
power used (lOx, 20x, etc.), type of preservative used, and a box for
the initials of the person who does the quality control check of the
multiplication and addition on the examination sheet.

5) Center of the phytoplankton examination sheet - seven algal classes
and eight types are delineated. Identifications are recorded under
the organism column, running counts are recorded under counts. The
running counts are tallied and multiplied by the S-R factor to obtain
totals in cells/ml. A total is given for each class and type as well
as for the sample.

Determination of the S-R Factor:

When strip counts or field counts are done on a Sedgwick-Raf ter counting
cell, only a portion of the 1 ml sample is examined. Therefore, a
calibration of "S-R" factor must be determined. The following formula
is used in this calibration:

S-R factor (strip count) = 10QQ mm3

LxWxDxS

where: L = length of a strip (mm)
S-R cell is 500 mm long

W = width of a strip which is the

Whipple grid image width (deter-
mined by using a stage micrometer)

D = depth of chamber (1 mm)

S = number of strips counted

The S-R (strip count) times C, the number of organisms counted (tally)
equals the number of algae per milliliter.

units/ml = S-R (strip count) x C

The S-R factor (field count) is calculated by using the following
formula:

S-R factor (field count) = 1QQQ Bm3

AxDxF

where: A = area of a field, which is the
Whipple grid image area

D = depth of chamber (1 mm)

F = number of field counts

27

The number of algae per ml equals the S-R (field count) times C, the
number of organisms counted (tally).

Units/ml = S-R (field count) x C

Procedure for Phytoplankton Counts :

In the unit (or clump) count each cell or colonial group of cells
receives one unit.

Examples :

1. Anacystis - one unit per clump

2. Anabaena - one unit per chain

3. "Filamentous green" - one unit per filament

4. Scenedesmus - one unit each (4, 8, 16 etc., celled organism.)

5. Fragilaria and Melosira - count each cell (may be best to average
the area for a single cell and divide into total area.)

6. Asterionella - each "arm" one unit

7. Dinobryon - each colony one unit.

An attempt is made to identify all organisms to generic level. If this
can not be accomplished then an effort is made to assign the organism to
the proper class and type. Unidentified organisms are described as "UI"
on the phytoplankton examination sheet. Subscripts are assigned, i.e.,
"UI1", "UI2", "UI3", etc., if more than one kind of unidentified
organism are present within a particular class and type.

Counts below 500 cells/ml are generally unreliable. In general, an
attempt is made to observe at least 20 organisms while making tallies in
strip counts. Any manipulation of the sample (concentration or dilution)
adds error. Therefore, on samples with high concentrations, a field
count rather than concentrate is performed. On samples with low counts,
more strips are counted. Precision is achieved in field counts by
determining the coefficient of variation for counts in the number of
fields counted and adjusting the number of fields counted to meet an +
10% error, as outlined in precision calculations (see: "Precision Data").

28

PHYTOPLANKTON

Precision Data:

N n-1
Where:

S = Standard deviation
M = Mean (average)
X = Count
n = Number of fields

2. Cv =

or

y 2

\J l!i

m

n-1

Where Cv = Coefficient of variation
3. P = % standard deviation of mean =

100c

v

\F

4. Cv must be 0.317 or less if results in a 10-field count are to be + 10%
within a 2/3 probability and a practical certainty (95%) of +_ 20% precision
error.

5. Using past data it was found that if ten fields are counted:

Cv = 1.0 or _+ 31.7% error was found in 90% of samples
0.7 or + 22% error was found in 75% of samples
0.45 or +_ 14% error was found in 50% of samples
0.317 or _+ 10% error was found in 33% of samples

On the average, a third of random samples were within +40% error when 10
fields were counted, and half were within _+14%.

6. P = The Standard error of count (percent) and is found in the log-log plot
of Cv versus n.

29

3.1.5 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories
_[ state vehicle, clipboard

J roof racks

J boat trailer

JJ pram, oars (and locks)

| canoe, paddles

3J boat, motor, gas can (and line)
J anchor, rope

life jackets, seat pads

Field Apparel

\_\ rain gear (jacket, pants, hat)
__J hip boots and/or chest waders
__j rubber gloves

Collecting and Sampling Gear

j secchi disk
_J pocket thermometer

[ photometer

tape measure
I j range finder

[ plastic bucket, rope
| plastic tubing with weight attached
J glass and/or plastic vials
J glass and/or plastic jars, bottles
sample preservative, fixative

Miscellaneous Items

| USGS topographic maps

: I clipboard

field data sheets, maps

_ tags and labels (with elastics
or string)

j pencils, pens

field identification manuals, keys

i J dissecting kit, hand lens

i | camera, film

;_ first-aid kit

field glasses

fj insect repellent

I I tool kit

[ cooler(s) , ice

30

PHYTOPLANKTON

3.1.6 DATA RECORD SHEETS

31

►J

o

as

H

Z

o

u

z

o

a

1-1

as

H

O

P 33

u

J O

w

-J z

as

o <

cu as

<:

03

H

ai

<3

fed CO

Q

H Cd

<d u

a

3 M

J

>

w

fe as

M

o w

tti

CO

z

z

O -J

o

M <C

H

CO U

^

i— i i— i

z

> z

<

>-< 3-

J

Q O

Q-,

W

o

co H

H

H

>H

H

33

cd

CU

CO

3

32

U

<d

CO

CO

z
o

I— I
H
a-
i— (
as
O
co
to
Q

C-l
<

M

<
33

..

O

as

• •

h- 1

Ed

Ed

H

>

H

<

t-i

M

H

aS

CO

CO

Oh

s

CxJ

H
i— i

CO

32

PHYTOPLANKTON

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

PHYTOPLANKTON EXAMINATION SHEET

River/Lake

Station

Dole Collected

Lot) No.

Analysis by-

Mi

Count

Date Analyzed

Closs

Type

Organism

Count

Tolly

Cells/ml

Total

*••

1

a
5

•
o
•

>■
e.
a.
o

o

'u
a

CQ

u

c
o

o

e

o

c
c

2 c

u at

>■ «-

c a

a. '
o 2

c -=

(J

■a

o
(J
o
o

o

HI

3

o

c
«

e

a

c
•

•

•
o
a
•j

O

w
O

V

o

u
o
o

-

e

VI
31

Q

O

c
<l

6
a

iZ

in

■ 01

Chrysophyceae
[ (Golden- Browns)

Cryptophyceae
(Cryptomonads)

Oinochyceae
(Dinoflaqeilates)

Euqienophyceoe
{Ejqlenids)

/~ki / / ^ Tot. live alaae (c
Chlorophyll a/ in mg/m-3 '

/ml)
SR =

Microscope Pnw^r Preserved

Qui

jlity CootrcH

33

3.1.7 REFERENCES

1. Collins, F.S. 1970. The Green Algae of North America. J. Cramer
Publisher, Lehke, Germany. Ill plates + 106 pp.

2. Edmondson, W.T., (ed.). 1959. Freshwater Biology. John Wiley and Sons,
Inc., New York, xxii + 1248 p.

3. Ettl, N. , J. Gerloff, and H. Heynig. 1978. Susswasser Flora von
Mitteleuropa Xanthophyceae. Part 1. Springer-Verlag, New York, xiv +
530 p.

4. Greenberg, A.E., R.R. Trussell, L.S. Clesceri, and M.H. Franson, (eds.).
1985. Standard Methods for the Examination of Water and Wastewater.
American Public Health Association, Washington, D.C. xlix + 1268 p.

5. Greeson, P.E. 1982. An Annotated Key to the Identification of Commonly
Occurring and Dominant Genera of Algae Observed in the Phytoplankton of
the United States. United States Geological Survey Water Supply Paper
2079. Superintendent of Documents, Washington, D.C. vi + 138 p.

6. Hansmann, E.W. 1973. Diatoms of the Streams of Eastern Connecticut.
Bulletin 106. State Geological and Natural History Survey of Connecticut,
Hartford. vi + 119 p.

7. Lamb, I.M., M.H. Zimmermann, and E.E. Webber. 1977. Artificial Key to
the Common Marine Algae of New England North of Cape Cod. Farlow
Herbarium, Harvard University, Cambridge, Massachusetts. 53 p.

8. Palmer, CM. 1977. Algae and Water Pollution - An Illustrated Manual
on the Identification, Significance, and Control of Algae in Water
Supplies and in Polluted Water. EPA-600/9-77-036. United States
Environmental Protection Agency, Municipal Environmental Research
Laboratory, Cincinnati, Ohio. viii + 124 p.

9. Prescott, G.W. 1968. The Algae: A Review. Houghton Mifflin Company,
Boston, xii + 436 p.

10. Prescott, G.W. 1970. How to Know the Freshwater Algae. Wm. C. Brown
Company, Publishers, Dubuque, Iowa. viii + 348 p.

11. Prescott, G.W. 1982. Algae of the Western Great Lakes. Otto Koeltz
Science Publishers, Koeningotein, West Germany. xiii + 977 p.

12. Rieth, A., J. Gerloff, and H. Heynig. 1980. Susswasser Flora von
Mitteleuropa Xanthophyceae. Part 2. Springer-Verlag, New York. vii +
147 p.

13. Shubert, L. 1984. Algae as Ecological Indicators. Academic Press,
London. xii + 434 p.

34

PHYTOPLANKTON

14. Slack, K.V., R.C. Averett, P.E. Greeson, and R.G. Lipscomb. 1973.
Methods for Collection and Analysis of Aquatic Biological and Micro-
biological Samples. Techniques of Water Resources Investigations of the
United States Geological Survey. Chapter A 4, Book 5 (Laboratory
Analysis). Superintendent of Documents, Washington, D.C. vi + 165 p.

15. Smith, G.M. 1950. Freshwater Algae of the United States. McGraw-Hill
Book Co., New York, vii + 719 p.

16. United States Environmental Protection Agency. 1980. Microscopic
Analysis of Activated Sludge. EPA/430/1-80-007. National Training and
Operational Technology Center, Cincinnati, Ohio. iv + 446 p.

17. United States Geological Survey. 1977. National Handbook of Recommended
Methods for Water-Data Acquisition. Office of Water Data Coordination,
Reston, Virginia. i + 741 p.

18. VanLandingham, S.L. 1982. Guide to the Identification, Environmental
Requirements and Pollution Tolerance of Freshwater Blue-Green Algae
(Cyanophyta) . EPA-600/3-82-073. U.S. EPA, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio, x + 341 p.

19. Vollenweider, R.A. , (ed.). 1974. A Manual on Methods for Measuring
Primary Production in Aquatic Environments. IBP Handbook No. 12.
Blackwell Scientific Publications, Oxford, England. xviii + 225 p.

20. Weber, C.I. 1971. A Guide to the Common Diatoms at Water Pollution
Surveillance System Stations. U.S. EPA, National Environmental Research
Center, Cincinnati, Ohio. iv + 101 p.

21. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA-670/4-73-
001. U.S. EPA, National Environmental Research Center, Cincinnati, OH.
xii + 146 p. + appendices.

35

SECTION

PAGE

3.0

BIOLOGICAL FIELD AND LABORATORY METHODS

3.2 Periphyton

3.2.1 Definition

3.2.2 Objectives

3.2.3 Field Sampling

3.2.4 Laboratory Analyses
Log-In Procedure
Microscopic Analysis

Periphyton Examination Laboratory Equipment List
3 2.5 Field Equipment and Supply List

3.2.6 Data Record Sheets

3.2.7 References

37
37
37
37
37
37
37
38
39
40
43

36

PERIPHYTON
3.2 PERIPHYTON

3.2.1 DEFINITION: Periphyton as used here shall mean the attached algal
community. Any associated bacteria, fungi, mosses or epiphytic animals
are identified to a rudimentary level only. Occasionally, planktonic
algae are collected during periphyton sampling in lotic waters. These
identifications are reported under the heading periphyton, although,
strictly speaking, they are plankton.

3.2.2 OBJECTIVES

1. To document the existing periphyton component in lotic and lentic
environments and determine dominant types; and

2. to evaluate water quality conditions by the use of indicator
species .

3.2.3 FIELD SAMPLING

Prior to disturbing the streambed or lakebed, stations (or reaches)
selected for qualitative investigation are visually inspected for algal
growth. Representative samples are collected from each macrohabitat :
pools, riffles, channel, streambank, backwater, open-water; and all
substrates: rocks, sand, vegetation, twigs and other debris. Each type
of alga encountered is collected using forceps, pipette, knife or by
hand. Specimens are placed in labeled glass (or plastic) vials with
water from the sampling site, and deposited into a cooler on ice for
transportation to the laboratory. Information concerning growth habit
and relative abundance of the representative algae are duly noted on
field sheets. Photographic documentation of site conditions may also be
conducted.

3.2.4 LABORATORY ANALYSES

Log-In Procedure

Each sample is recorded in the algae log book along with the phytoplank-
ton samples. The sample is assigned a number followed by the letter P
indicating periphyton. This is done in order to distinguish it from
phytoplankton samples. Also recorded in the log book are the station
number and location, the date collected, initials of the collector, and
date analyzed.

Microscopic Analysis

Samples collected in the field are stored in the refrigerator until they
are viewed. Specimens are identified within one to two days following
collection while they are still alive and healthy. This facilitates
identification since preservatives tend to alter the color and - in some
cases - the structure of the algae. Identifications are made from wet
mounts using a compound microscope equipped with lOx, 20x, 40x, and lOOx
objectives. Identifications are made using various taxonomic keys to the
lowest level possible and recorded on a Periphyton Lab Bench Sheet (see
Section 3.2.6). Certain specimens are photographed as a means of docu-
mentation and for use in presentations or as a teaching tool. Taxonomic
lists of the results are compiled for each survey and published in
appropriate reports.

37

Periphyton Examination Laboratory Equipment List

1. Microscope with lOx, 20x, 40x, lOOx objectives

2. Microscope slides and coverslips

3. Pipettes

4. Forceps, probes

5 . Lens paper

6. Bench sheets

38

PERIPHYTON

3.2.5 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories

j | state vehicle, clipboard

; | roof racks

|~j boat trailer

j | pram, oars (and locks)

|~| canoe, paddles

{ | boat motor, gas can (and line)

j | anchor, rope

1 | life jackets, seat pads

Collecting and Sampling Gear

j [ secchi disk

j [ pocket thermometer
j I photometer
| j tape measure

| [ range finder

|~j plastic bucket, rope

1 i glass and/or plastic vials

j [ glass and/or plastic jars, bottles

■ [ sample preservative, fixative

Field Apparel

| | rain gear (jacket, pants, hat)

j [ hip boots and/or chest waders

I J rubber gloves

Miscellaneous Items

| j USGS topographic maps

| { clipboard

j j field data sheets, maps

| i tags and labels (with elastics or
string)

I [ pencils, pens

| | field identification manuals, keys

J J dissecting kit, hand lens

j ( camera, film

[~~j first-aid kit

J~ J field glasses

) j insect repellent

Q tool kit

| | cooler(s) , ice

39

3.2.6 DATA RECORD SHEETS

40

PERIPHYTON

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MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

PERIPHYTON LAB BENCH SHEET

BASIN:
RIVER:
STATION:

BASIN NUMBER:

STREAM INVENTORY NUMBER:

COMMENTS

TOWN:

Sample #:

Habitat

Date Collected:

Date Analyzed:

Collector(s) :

Analysis by:

Microscope: Power:

Number of Samples:

Photo:

Substrate

Relative
Abundance

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Code(s) :

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Habitat

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Relative
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Identification:

Code(s)

Sample #:

Habitat

Substrate:

Relative
Abundance

Identification:

Code(s)

Relative Abundance: Most Abundant, Abundant, Common, Sparse
Habitat: Pool, Riffle, Backwater, Impoundment, Spillway, etc
Substrate: Rock, Mud, Sand, Wood, Bottle, etc.

42

PERIPHYTON

3.2.7 REFERENCES

1. Collins, F.S. 1970. The Green Algae of North America. J. Cramer
Publisher. Lehre, Germany. 106 p.

2. Greenberg, A.E., R.R. Trussell, L.S. Clesceri, and M.H. Franson, (eds.).
1985. Standard Methods for the Examination of Water and Wastewater.
American Public Health Association, Washington, D.C. xlix + 1268 p.

3. Edmondson, W.T. and G.G. Winberg (eds.). 1971. A Manual on Methods for
the Assessment of Secondary Productivity in Fresh Waters. IBP Handbook
No. 17. Blackwell Scientific Publications, Oxford, England. xxiv +
358 p.

4. Hansman, E.W. 1973. Diatoms of the Streams of Eastern Connecticut.
Bulletin 106. State Geological and Natural History Survey of
Connecticut, Hartford, vi + 119 p.

5. Hynes, H.B.N. 1970. The Ecology of Running Waters. University of
Toronto Press, Ontario, Canada. xxiv + 555 p.

6. Hynes, H.B.N. 1974. The Biology of Polluted Waters. University of
Toronto Press, Ontario, Canada. xiv + 202 p.

7. Patrick, R. , and C.W.Reimer. 1966. The Diatoms of the United States.
Volume I. Philadelphia Academy of Natural Sciences, Philadelphia. xi +
688 p.

8. Patrick, R. , and C.W. Reimer. 1975. The Diatoms of the United States.
Volume II. Philadelphia Academy of Natural Sciences, Philadelphia. ix
+ 213 p.

9. Prescott, G.W. 1982. Algae of the Western Great Lakes Area. Wm. C.
Brown Co., Dubuque. xiii + 977 p.

10. Prescott, G.W. 1978. How to Know the Freshwater Algae. Wm. C. Brown
Co., Dubuque. x + 293 p.

11. Smith, G.M. 1950. The Freshwater Algae of the United States. McGraw
Hill Book Co., Inc., New York. vii + 719 p.

12. Shubert, L. 1984. Algae as Ecological Indicators. Academic Press,
London. xii + 434 p.

13. VanLandingham, S.L. 1982. Guide to the Identification, Environmental
Requirements and Pollution Tolerance of Freshwater Blue-Green Algae
(Cyanophyta). EPA-600/3-82-073. United States Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati.

ix + 341 p.

14. Vollenweider, R.A. , (ed.). 1974. A Manual on Methods for Measuring
Primary Production in Aquatic Environments. IBP Handbook No. 12.
Blackwell Scientific Publications, Oxford, England. xviii + 225 p.

43

15. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA-670/4-73-001
United States Environmental Protection Agency, National Environmental
Research Center, Cincinnati, Ohio, xii + 146 p. + appendices.

16. Vinyard, W.C. Diatoms of North America.
Eureka, CA. 119 p.

1979. Mad River Press, Inc.,

44

AQUATIC AND WETLAND VEGETATION

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.3 Aquatic and Wetland Vegetation 46

3.3.1 Definition 46

3.3.2 Objectives 46

3.3.3 Field Sampling 46

3.3.4 Laboratory Analyses 46

3.3.5 Field Equipment and Supply List 47

3.3.6 Data Record Sheets 48

3.3.7 References 51

45

3.3 AQUATIC AND WETLAND VEGETATION

3.3.1 DEFINITION: Aquatic flora as used here pertains to several taxonomic
groups including the Characeae (stoneworts and muskgrass); Musci,
Hepaticae, and Ricciaceae (mosses, leafy liverworts, thallose
liverworts); Osraundaceae (flowering ferns); Equisetaceae (horsetail,
scouring rush); Isoetaceae (Quillwort); and the Angiospermae (the seed
plants) .

3.3.2 OBJECTIVES

1. To identify and test reliable methods and procedures for the
collection, identification and enumeration of aquatic and wetland
vegetation;

2. to document existing aquatic plant species and communities; and

3. to determine areal coverage and dominant plant types.

3.3.3 FIELD SAMPLING

For riverine habitats, aquatic and wetland vegetation are located and
qualitatively mapped by visually examining the streambed, streamside,
and immediate riparian areas by walking or wading. A reach of stream
approximately 10-meters in length is generally investigated. Each
macrohabitat is sampled and the predominant vegetation noted and
recorded on standard type field data sheets. A schematic map is pre-
pared for each site. Photographic documentation is sometimes made.
Vegetation is generally identified on-site.

The aquatic and wetland plant community in lacustrine habitats is
located and mapped by examining the limnetic, shoreline, and littoral
areas by boat or waders. Occasional samples are collected at regular
intervals on imaginary transects run across open-water areas of the
lake or impoundment. All habitats are sampled and the relative
abundance of each plant type noted and mapped on prepared outline maps.
Representative macrophytes are collected by hand and, in deeper water,
by dragging a simple grappling hook with a weight attached to the shaft.
An Ekman or Ponar dredge is sometimes used to collect deeply-submerged
vegetation. Identifications of most plant specimens are made in the
field.

3.3.4 LABORATORY ANALYSES

Vegetation not identified in the field is collected and returned to the
laboratory for further analysis using a stereoscopic microscope or hand
lens and various taxonomic keys. Representative plant specimens
collected from each site are pressed and dried in preparation for
permanent mounting. Plant specimens are deposited in the Botanical
Reference Library of the Technical Services Branch.

46

3.3.5 FIELD EQUIPMENT AND SUPPLY LIST

AQUATIC AND WETLAND VEGETATION

Vehicles, Boats and Accessories

j | state vehicle, clipboard

I I roof racks

j~[ boat trailer

f~] pram, oars (and locks)

j J canoe, paddles

j \ boat motor, gas can, (and line)

j~~j anchor, rope

j| life jackets, seat pads

Collecting and Sampling Gear

j [ secchi disk

j [ pocket thermometer

j] photometer

j | tape measure

j | range finder

| I plastic bucket, rope

j j glass and/or plastic vials

j [ glass and/or plastic jars, bottles

j~~| plastic bags (and ties)

[ [ sample preservative, fixative

I j rake

1 [ grappling hook, rope

j [ Ekman, Ponar dredges

|_J white enamel trays

[ | trowel

| plant press and vasculi

Field Apparel

I [ rain gear, (jacket, pants, hat)

J hip boots and/or chest waders

j I rubber gloves

Miscellaneous Items

[~~| USGS topographic maps

_] clipboard

| I field data sheets, maps

! | tags and labels (with elastics or

string)

j [ pencils, pens

| | field identification manuals, keys

| [ dissecting kit, hand lens

[J first-aid kit

| \ field glasses

[_J insect repellent

| [ tool kit

| [ cooler(s) , ice

.urn

47

3.3.6 DATA RECORD SHEETS

48

AQUATIC AND WETLAND VEGETATION

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AQUATIC AND WETLAND VEGETATION

!

3.3.7 REFERENCES

1. Beal, E.O. 1977. A Manual of Marsh and Aquatic Vascular Plants of North
Carolina with Habitat Data. Technical Bulletin Number 247. North
Carolina Agricultural Experiment Station, Raleigh. iv + 298 p.

2. Burkhalter, A. P., L.M. Curtis, R.L. Lazor, M.L. Beach, and J.C. Hudson.
1977. Aquatic Weed Identification and Control Manual. Florida
Department of Natural Resources, Bureau of Aquatic Plant Research and
Control, Tallahassee, vii + 100 p.

3. Commonwealth of Pennsylvania. 1971. Aquatic Plants: A Guide for Their
Identification and Control in Pennsylvania. Pennsylvania Water Resources
Coordinating Committee, Harrisburg. ii + 63 p.

4. Conard, H.S. 1975. How to Know the Mosses and Liverworts. W.C. Brown
Company Publishers, Dubuque, Iowa. x + 226 p.

5. Correll, D.S. and H.B. Correll. 1972. Aquatic and Wetland Plants of
Southwestern United States. United States Environmental Protection
Agency, Washington. xv + 1777 p.

6. Crow, G.E. and C.B. Hellquist. 1981. Aquatic Vascular Plants of New
England: Part 2. Typhaceae and Sparganiaceae. Bulletin 517. New
Hampshire Agricultural Experiment Station, University of New Hampshire,
Durham. ii + 21 p.

7. Crow, G.E. and C.B. Hellquist. 1982. Aquatic Vascular Plants of New
England: Part 4. Juncaginaceae , Scheuchzeriaceae , Butomaceae,
Hydrocharitaceae . Bulletin 520. New Hampshire Agricultural Experiment
Station, University of New Hampshire, Durham. ii + 20 p.

8. Crow, G.E. and C.B. Hellquist. 1983. Aquatic Vascular Plants of New
England: Part 6. Trapaceae, Haloragaceae , Hippuridaceae. Bulletin 524.
New Hampshire Agricultural Experiment Station, University of New
Hampshire, Durham. ii + 26 p.

9. Crow, G.E. and C.B. Hellquist. 1985. Aquatic Vascular Plants of New
England: Part 8. Lentibulariaceae. Bulletin 528. New Hampshire
Agricultural Experiment Station, University of New Hampshire, Durham,
ii + 22 p.

10. Dennis, W.M. and B.G. Isom, (eds.). 1984. Ecological Assessment of
Macrophyton: Collection, Use and Meaning of Data. ASTM Special
Technical Publication 843. American Society for Testing and Materials,
Philadelphia. ix + 122 p.

11. Edmondson, W.T. and G.G. Winberg, (eds.). 1971. A Manual on Methods for
the Assessment of Secondary Productivity in Fresh Waters. IBP Handbook
No. 17. Blackwell Scientific Publications, Oxford, England. xxiv +

358 p.

51

_

12. Fairbrothers, D.E., E.T. Moul, A.R. Essback, D.N. Riemer, and D.A.
Schallock. (Not dated). Aquatic Vegetation of New Jersey. Part I:
Ecology and Identification; and Part II: Problems and Methods of Control,
Extension Bulletin 382. Extension Service, College of Agriculture,
Rutgers - The State University, New Brunswick, New Jersey. 107 p.

13. Fassett, N.C. 1972. A Manual of Aquatic Plants (with revision appendix
by E.C. Ogden). University of Wisconsin Press, Madison. ix + 405 p.

14. Faust, M.E. 1977. Field Guide to the Grasses, Sedges, and Rushes of the
United States. Dover Publications, Inc., New York. 83 p.

15. Godfrey, R.K. and J.W. Wooten. 1979. Aquatic and Wetland Plants of
Southeastern United States: Monocotyledons. University of Georgia
Press, Athens. ix + 712 p.

16. Godfrey, R.K. and J.W. Wooten. 1981. Aquatic and Wetland Plants of
Southeastern United States: Dicotyledons. University of Georgia Press,
Athens. ix + 933 p.

17. Hellquist, C.B. and G.E. Crow. 1980. Aquatic Vascular Plants of New
England: Part I. Zosteraceae, Potamogetoraceae , Zannichelliaceae,
Najadaceae. Bulletin 515. New Hampshire Agricultural Experiment Station,
University of New Hampshire, Durham. iii + 68 p.

18. Hellquist, C.B. and G.E. Crow. 1981. Aquatic Vascular Plants of New
England: Part 3. Alismataceae. Bulletin 518. New Hampshire
Agricultural Experiment Station, University of New Hampshire, Durham,
iii + 32 p.

19. Hellquist, C.B. and G.S.. Crow. 1982. Aquatic Vascular Plants of New
England: Part 5. Araceae, Leranaceae, Xyridaceae, Eriocaulaceae, and
Pontederiaceae. Bulletin 523. New Hampshire Agricultural Experiment
Station, University of New Hampshire, Durham. iii + 46 p.

20. Hellquist, C.B. and G.E. Crow. 1984. Aquatic Vascular Plants of New
England: Part 7. Cabombaceae, Nymphaeaceae, Nelumbonaceae, and
Ceratophyllaceae. Bulletin 527. New Hampshire Agricultural Experiment
Station, University of New Hampshire, Durham. ii + 27 p„

21. Hotchkiss, N. 1972. Common Marsh, Underwater, and Floating-Leaved
Plants of the United States and Canada. Dover Publicatons, Inc., New
York, v + vii + 233 p.

22. Knudsen, J.W. 1966. Collecting and Preserving Plants and Animals.
Harper and Row Publishers, New York. x + 320 p.

23. Mager, D.W. 1981. Freshwater Wetlands: A Guide to Common Indicator
Plants of the Northeast. University of Massachusetts Press, Amherst,
ix + 246 p.

24. Muenscher, W.C. 1944. Aquatic Plants of the United States. Cornell
University Press, Ithaca. ix + 374 p.

52

AQUATIC AND WETLAND VEGETATION

25. Ogden, E.C. 1974. Potamogeton in New York. Bulletin 423. New York
State Museum and Science Service, Albany, v + 20 p.

26. Ogden, E.C. 1974. Anatomical Patterns of Some Aquatic Vascular Plants
of New York. Bulletin 424. New York State Museum and Science Service,
Albany, v + 133 p.

27. Prescott, G.W. 1969. How to Know the Aquatic Plants. W.C. Brown, Co.,
Dubuque. viii + 171 p.

28. Schwoenbel, J. 1970. Methods of Hydrobiology (Freshwater Biology).
Pergamon Press, Inc., Elmsford, New York, x + 200 p.

29. Seymour, F.C. 1969. The Flora of New England. C.E. Tuttle Co.,
Rutland, Vermont. xvi + 596 p.

30. Steward, A.N. , L.R.J. Dennis, and H.M. Gilkey. 1960. Aquatic Plants of
the Pacific Northwest with Vegetative Key*s . Studies in Botany Number 11,
Oregon State College, Corvallis. vi + 184 p.

31. United States Array Corps of Engineers. 1977. Wetland Plants of the
Eastern United States. North Atlantic Division, New York. 126 p.

32. United States Array Corps of Engineers. 1979. Wetland Plants of the
Eastern United States (Supplement 1). North Atlantic Division, New York,
210 p.

33. United States Environmental Protection Agency. 1982. New England
Wetlands: Plant Identification and Protective Laws. Region I, Boston.
168 p.

34. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA-670/4-73-001 .
U.S. Environmental Protection Agency, National Environmental Research
Center, Cincinnati, Ohio, xii + 146 p. + appendices.

35. Weldon, L.W. , R.D. Blackburn, and D.S. Harrison. 1973. Common Aquatic
Weeds. Dover Publications, Inc., New York. iv + 43 p.

36. Winterringer, G.S. and A.C. Lopinot. 1977. Aquatic Plants of Illinois.
Illinois State Museum Popular Science Series Volume VI. Illinois State
Museum, Springfield. 142 p.

53

SECTION

PAGE

3.0

BIOLOGICAL FIELD AND LABORATORY METHODS

3.4 Aquatic Macroinvertebrates

3.4.1 Definition

3.4.2 Objectives

3.4.3 Field Sampling
Qualitative
Rapid Assessment
Quantitative •

3.4.4 Laboratory Analyses

3.4.5 Field Equipment and Supply List

3.4.6 Data Record Sheets

3.4.7 References

55
55
55
55
55
55
56
56
58
59
63

54

AQUATIC MACRO INVERTEBRATES

3.4 AQUATIC MACROINVERTEBRATES

3.4.1 DEFINITION: The aquatic macroinvertebrate community is defined as the
assemblage of invertebrate organisms which can be seen by the unaided
eye and retained by a U.S. Standard No. 30 sieve (i.e., 28 meshes per
inch; 0.595 mm apertures). All or some life-cycle stages of these
animals occur either attached to plants, other animals, debris, or
inorganic substrates, or they float or swim in the water column of lentic
and lotic waterbodies. Representative members of this community include •
but are not limited to - sponges, bryozoa, flat worms, segmented worms,
arthropods (water mites, crustaceans, insects), and mollusks.

3.4.2 OBJECTIVES

1. To provide information for stream classification, assessment of water
quality conditions and trends, and direct impact assessment;

2. to interpret data using knowledge of the pollution ecology of
component taxa (e.g., indicator schemes; biotic indices), or by
observing changes in invertebrate community structure (e.g.,
richness; diversity); and

3. to determine the severity of water pollution problems by comparing
unimpacted control or reference communities with potentially
impacted communities.

3.4.3 FIELD SAMPLING

Qualitative

Qualitative macroinvertebrate sampling for stream classifications or
special site assessments involves the use of a variety of sampling
devices to collect samples from all available habitats encountered within
a sampling site. Generally, D-frame nets are used to sweep aquatic
vegetation, collect under cut stream banks, and agitate substrates to
dislodge benthic organisms. Depending upon the taxonomic level desired,
organisms are identified in the field to family level or placed in jars
with 70% ethanol (95% if sample contains sediment materials or debris)
for transport to the laboratory where further analyses are conducted.

Rapid Assessment

To obtain a sample for the Rapid Assessment Methodology, a D-frame net is
pressed against the substrates, and substrate material just upstream and
in front of the net is agitated by kicking. This procedure is continued
for five minutes while gradually moving upstream. Sampling is executed
in areas of comparable substrate and current velocity (usually riffle
areas within the central one third of the channel).

At the end of the five minutes of kick-sampling the contents of the net
are emptied into a white enamel pan. Organisms clinging to the net are
removed, using forceps, and placed in the sample container, as are

55

organisms on substrate materials too large to fit into the sample
container. Once the organisms have been removed, these larger materials
are returned to the stream. The remainder of the sample is added to the
container and preserved with 95% ethanol, containing 130 mg/1 Rose Bengal
stain. Completed labels are placed inside each container and attached
to the outside. Field notes record the major taxonomic groups
encountered during field processing.

Quantitative

When quantitative sampling is required, the following routine is
employed:

1. Depending on depth, flow, and substrate conditions sampling gear is
selected from among Ekman, Petersen and Ponar grab samplers or
Surber and Hess substrate samplers. One set of four replicate
samples is obtained following a random transect whereby both banks
and two quarter points are sampled.

2. The substrate obtained is characterized according to particle size
and composition, placed into a basin, and mixed thoroughly. When
the sample consists of heavily organic or sand-silt type substrate,
one-quarter of the sample is randomly selected and retained after
mixing. The remaining material is qualitatively examined and
discarded. Subsampling is often necessary due to the time required
for sorting a large quantity of substrate.

3. The sample portion is passed through a standard U.S. No. 30 brass
sieve (0.595 mm apertures). Organisms and substrate left behind are
placed into labeled plastic or glass wide-mouth containers (approx.

1 liter) and returned alive or preserved with 95% ethanol to the
laboratory for further analysis.

3.4.4 LABORATORY ANALYSES

All samples are recorded in a log book upon arrival at the laboratory.
Preserved samples are drained on a U.S. Standard No. 30 mesh screen and
rinsed with tap water. Live and preserved samples are placed in
individual white enamel pans for sorting. Samples for quantitative
analyses are preferably sorted alive by removing all benthic organisms
manually from the substrate and separating them by taxonomic order into
glass vials containing 70% ethanol. For the Rapid Assessment Methodo-
logy, the contents of the enamel pan are subdivided by scooping material
successively (one after the other) into four to eight glass petri dishes
until all the material is distributed among the dishes. The number of
dishes used depends on the volume of substrate and debris in the sample.

Before picking out organisms, the petri dishes are assigned a number (one
to four, if four are used). Numbers are then drawn at random to deter-
mine the order of processing. The dish with the number corresponding to
the first number drawn is placed on the stage of a stereomicroscope by
deliberate orientation (first random field). All organisms within the
field of view at low power are picked and placed in labeled vials with
70% ethanol. When all organisms in the field of view have been removed

56

AQUATIC MACROINVERTEBRATES

the dish is moved Co another random field for removal of additional
organisms. This procedure is repeated until 100 organisms have been
selected, moving to the next randomly selected petri dish as required.
The remaining sample materials are again sieved on a #30 mesh screen,
labeled, and archived in 95% ethanol.

Macroinvertebrate specimens other than chironomids and oligochaetes are
identified through examinations using a Wild M5A stereomicroscope
equipped with fiber optics lighting. Oligochaetes, chironomid larvae,
and chironomid pupae must be mounted on microscope slides before
examination with an Olympus BH-2 compound microscope equipped with
Nomarski optics. Semi-permanent slide mounts are made by placing the
specimens on a 25 x 75 mm microscope slide in CMC-10. The oligochaetes
and chironomid larvae are mounted in the CMC-10 without prior clearing.
The heads of the chironomid larvae are excised and positioned above the
bodies, usually with three specimens under each of two 18 x 18 mm square
coverslips per slide. Chironomid pupae are first cleared in 10% KOH
(potassium hydroxide) before mounting in CMC-10 with one specimen per
18 x 18 mm slide. The heads of the pupae are also separated from the
body once mounted on the slide.

57

3.4.5 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories

I \ state vehicle, clipboard

j J roof racks

[ [ boat trailer

[ I pram, oars (and locks)

j canoe, paddles

f~J boat motor, gas can (and line)

| [ anchor, rope

| j life jackets, seat pads

Collecting and Sampling Gear
j J pocket thermometer
| ; tape measure

I j range finder

| _J Ekman, Peterson, Ponar dredges

j { Surber samplers

j [ Hess sampler

[~] metal holding tub

| J white enamel trays

j J sieves (of various sizes)

j I plastic bucket, rope

; _] glass and/or plastic vials

| J glass and/or plastic jars, bottles

j j ethanol, formalin

| J killing jar, killing agent

[_] aerial net, D-frarae net

Field Apparel

! | rain gear (jacket, pants, hat)

j j hip boots and/or chest waders
I [ rubber gloves

Miscellaneous Items

, [ USGS topographic maps

|_J clipboard

! field data sheets, maps

I | tags and labels (with elastics or
string)

[ pencils , pens

| | field identification manuals, keys

j [ dissecting kit, hand lens

j j camera, film

| | first-aid kit

) | field glasses

| j insect repellent

! | tool kit

1 j cooler(s) , ice

58

AQUATIC MACROINVERTEBRATES

3.4.6 DATA RECORD SHEETS

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60

AQUATIC MACRO INVERTEBRATES

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

AQUATIC MACRO INVERTEBRATE LAB BENCH SHEET

Name of Water Body
Date Collected

Station No.

Location

Collector

Sorted By

Code

********************************************************************

ORGANISM # /LS/TV/TI | ORGANISM # /LS/TV/TI

*******************************************************************************

Nematoda

Plecoptera

Annelida

Oligochaeta
Hirudinea

Hemiptera

Megaloptera

Isopoda

Amphipoda
Decapoda

Trichoptera

Coleoptera

Hydracarina

Diptera

Collembola

Ephemeroptera

Gastropoda
Pelecypoda

Odonata

Others

*******************************************************************************

Total No. of Organisms
Total No. of Kinds

# = Number of individuals tallied
TV = Biotic Index Tolerance Value
TI = Taxonoraist's initials
LS = Life stage: I = Immature

P = Pupa
A = Adult

£i

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

SLIDE INVENTORY CATALOG SHEET

Page

of

SURVEY NAME:

SURVEY CODE

SLIDE BOX OF

SLOT /STATION CS TAXA

COMMENTS

SLOT /STATION CS TAXA COMMENTS

B

B

B

B

B

B

B

B

B

B

CS = cover slip

62

AQUATIC MACROINVERTEBRATES

3.4.7 REFERENCES
General

1. Edmondson, W.T. and G.G. Winberg, (eds.). 1971. A Manual on Methods for
the Assessment of Secondary Productivity in Fresh Waters. IBP Handbook
No. 17. Blackwell Scientific Publications, Oxford, England. xxiv +

358 p.

2. Green, R.H. 1979. Sampling Design and Statistical Methods for Environ-
mental Biologists. John Wiley and Sons, New York, xiv + 257 p.

3. Hilsenhoff, W.L. 1982. Using a Biotic Index to Evaluate Water Quality
in Streams. Technical Bulletin No. 132. Wisconsin Department of Natural
Resources, Madison. 22 p.

4. Schwoerbel, J. 1970. Methods of Hydrobiologj* (Freshwater Biology).
Pergamon Press, Inc., Elrasford, New York. x + 200 p.

5. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. . EPA-670/4-73-001 .
United States Environmental Protection Agency, National Environmental
Research Center, Cincinnati, Ohio. xii + 146 p. + appendices.

Taxonomic

1. Allen, R.K. and G.F. Edmonds, Jr. 1959. A Revision of the Genus
Ephemerella (Ephemeroptera: Ephemerellidae) . I. The Subgenus
Timpanoga. The Canadian Entomologist. 91: 51-58.

2. . 1961. A Revision of the Genus Ephemerella

(Ephemeroptera: Ephemerellidae). II. The Subgenus Caudatella. Annals
Entomol. Soc. Amer. 54: 603-612.

3. . 1961. A Revision of the Genus Ephemerella

(Ephemeroptera: Ephemerellidae) III. The Subgenus Attenuattella.
Journal of Kansas Entomological Society 34(4): 161-173.

4. . 1962. A Revision of the Genus Ephemerella

(Ephemeroptera: Ephemerillidae) IV. The Subgenus Dannella. Journal of
Kansas Entomological Society. 35(3): 333-338.

5. . 1962. A Revision of the Genus Ephemerella

(Ephemeroptera: Ephemerillidae) V. The Subgenus Prunella in North
America. Misc. Publ. Entomol. Soc. Amer. 3(5): 147-179.

6. . 1963. A Revision of the Genus Ephemerella

(Ephemeroptera: Ephemerillidae) VI. The Subgenus Serratella in North
America. Ann. Ent. Soc. Amer. 56: 583-600.

7. . 1963. A Revision of the Genus Ephemerella

(Ephemeroptera: Ephemerillidae) VII. The Subgenus Eurylophella. The
Canadian Entomologist. 95: 597-623.

63

8. . 1965. A Revision of the Genus Ephemerella
(Ephemeroptera: Ephemeriilidae) VIII. The Subgenus Ephemerella in North
America. Misc. Publ. Encomol. Soc. Amer. 6(4): 243-282.

9. Bednarik, A.F. and W.P. McCafferty. 1979. Brosystematic Revision of the
Genus Scenonema (Ephemeroptera: Heptageniidae) . The Canadian Bulletins
of Fisheries and Aquatic Sciences. No. 201. 73 p.

10. Bergman, E.A., W.L. Hilsenhoff. 1978. Baetis (Ephemeroptera: Baetidae)
of Wisconsin. The Great Lakes Entomologist. 11(3): 125-135.

11. Bode, R.W. 1983. Larvae of North American Eukief feriella and Tvetenia
(Diptera: Chironomidae) . New York State Museum, Albany. 40 p.

12. Brinkhurst, R.O., B.G.M. Jamieson. 1971. Aquatic Oligochaeta of the
World. University of Toronto Press, Toronto, Ontario, Canada. xii +
860 p.

13. Burch, J.B. 1972. Freshwater Spliaeriacean Clams (Mollusca: Pelecypoda)
of North America. U.S. Environmental Protection Agency, Washington, D.C.
vi + 31 p.

14. Edmunds, G.F., S.L. Jensen, and L. Berner.. 1976. The Mayflies of North
and Central America. Univ. Minnesota Press, Minneapolis. x + 330 p.

15. Hilsenhoff, W.L. 1982. Aquatic Insects of Wisconsin. University of
Wisconsin, Madison. 60 p.

16. Hiltunen, J.K. and D.J. Klemm. 1980. A Guide to the Naididae (Annelida:
Clitellata: Oligochaeta) of North America. EPA-600/4-80-031. U.S. EPA,
Cincinnati, Ohio. ix + 48 p.

17. Hobbs, H.H. 1972. Crayfishes (Astacidae) of North and Middle America.
U.S. EPA, Washington. x + 173 p.

18. Holsinger, J.R. 1972. The Freshwater Amphipod Crustaceans (Gammaridae)
of North America. U.S. EPA, Washington. viii + 89 p.

19. Hynes, H.B.N. 1970. The Ecology of Running Waters. Liverpool
University Press, Liverpool, England. xxiv + 555 p.

20. Jokinen, E.H. 1983. The Freshwater Snails of Connecticut. Bulletin
109. Connecticut Geological and Natural History Survey, Hartford,
vii + 83 p.

21. Klemm, D.J. 1982. Leeches (Annelida: Hirudinea) of North America.
EPA-600/3-82-025. U.S. EPA, Cincinnati, Ohio. xvii + 177 p.

22. Malcolm, S.E. 1971. The Water Beetles of Maine: Including the Families
Gyrinidae, Haliphidae, Dytiscidae, Noteridae, and Hydrophilidae.
Technical Bulletin 48. Life Sciences and Agriculture Experiment Station,
Orono, Maine. 49 p.

23. Maschwitz, D.E. 1975. Revision of the Nearctic Species of the Subgenus
Polypedilum (Chironomidae: Diptera). Ph.D. Thesis. Univ. of Minnesota,
Minneapolis. 325 p.

64

AQUATIC MACROINVERTEBRATES

24. Merritt, R.W. and K.W. Cummins, (eds.). 1984. An Introduction to the
Aquatic Insects of North America. Kendall/Hunt Publ. Co., Dubuque,
Iowa. ix + 722 p.

25. Oliver, D.R. and R.W. Bode. 1985. Description of the Larva and Pupa of
Cardiocladius albiplumus Saether (Diptera: Chironomidae) . The Canadian
Entomologist. 117(7): 803-809.

26. Oliver, D.R. and M.E. Roussel. 1983. The Genera of Laval Midges of
Canada. Minister of Supply and Services Canada, Ottawa, Ontario, Canada.
263 p.

27. Pennak, R.W. 1978. Fresh-water Invertebrates of the United States.
John Wiley & Sons, New York. xvii + 803 p.

28. Roback, S.S. 1981. The Immature Chironomids of the Eastern United
States V. Pentaneurini - Thienemannimyia Group. Proc. of the Academy of
Natural Sciences of Philadelphia. 133: 73-128. ~ ~

29. Ross, H.H. 1972. The Caddisflies, or Trichoptera, of Illinois.
Entomological Reprint Specialists, Los Angeles. 326 p.

30. Schefter, P.W. and G.B. Wiggins. 1986. A Systematic Study of the
Nearctic Larvae of the Hydropsyche morosa Group (Trichoptera:
Hydropsychidae) . Royal Ontario Museum Miscellaneous Publication,
Toronto. 94 p.

31. Schuster, G.A. , D.A. Ethier. 1978. A. Manual for the Identification of
the Larvae of the Caddisfly General Hydropsyche Pictet and Symphitopsyche
Ulmer in Eastern and Central North America (Trichoptera: Hydropsychidae).
EPA-600/4-78-060. U.S. EPA, Cincinnati, xii + 129 p.

32. Soponis, A.R. 1977. A Revision of the Nearctic Species of Orthocladius
(Orthocladius) van der Wulp (Diptera: Chironomidae). The Entomological
Society of Canada, Ottawa. 187 p.

33. Simpson, K.W. and R.W. Bode. 1980. Common Larvae of Chironomidae
(Diptera) from New York State Streams and Rivers. Bulletin No. 439.
New York State Museum, Albany, vi + 105 p.

34. Simpson, K.W. , R.W. Bode and P. Albu. 1983. Keys for the Genus
Cricotopus Adapted from "Revision der Gatting Cricotopus van der Wulp
and ihrer Verwandten (Diptera, Chironomidae)" by M. Hirvenoja. Bulletin
No. 450. New York State Museum, Albany, vi + 133 p.

35. Stimpson, K.S., D.J. Kleram, and J.K. Hiltunen. 1982. A Guide to the
Freshwater Tubificidae (Annelida: Clitellata: Oligochaeta) of North
America. EPA-600/4-80-031 . U.S. EPA, Cincinnati, x + 61 p.

36. Stone, A. 1964. Guide to the Insects of Connecticut, Part VI. The
Diptera or True Flies of Connecticut (Ninth Fasicle). Bulletin No. 97,
Connecticut Geological and Natural History Survey, Hartford, vii +
126 p.

65

37. Usinger, R.L., (ed.). 1956. Aquatic Insects of California. University
of California Press, Berkeley. x + 508 p.

38. Walker, E.M. 1953. The Odonata of Canada and Alaska, Volume One.
University of Toronto Press, Toronto, Canada, xi + 292 p.

39. . 1958. The Odonata of Canada and Alaska, Volume Two.

Univ. Toronto Press, Toronto, xi + 318 p.

40. Walker, E.M. and P.S. Corbet. 1975. The Odonata of Canada and Alaska,
Volume Three. Univ. Toronto Press, Toronto, xvi + 307 p.

41. Wiggins, G.B. 1977. Larvae of the North American Caddisfly
(Trichoptera) . University of Toronto Press, Toronto, xi + 401 p.

42. Williams, W.D. 1972. Freshwater Isopods (Asellidae) of North America.
U.S. EPA, Washington. ix + 45 p.

43. Wilson, R.S., J.D. McGill. 1982. A Practical Key to the Genera of Pupal
Exuviae of the British Chironomidae (Diptera, Insecta). University of
Bristol, Bristol, England. 62 p.

66

FISH

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.5 Fish 68

3.5.1 Definition 68

3.5.2 Objectives 68

3.5.3 Field Sampling 68
Physical Measurements 68
Gill Netting 68

"Electrofishing 69

Trapping 69

Processing 69

3.5.4 Laboratory Analysis 69
Processing 69
Aging 70

3.5.5 Data Management 70
Reporting of Results 70
Computer Files 70

3.5.6 Field Equipment and Supply List 71

3.5.7 Data Record Sheets and Freshwater and Anadromous Fishes

Coding List 72

3.5.8 References 80

67

3.5 FISH

3.5.1 DEFINITION: For the purpose of this standard operating procedure, fish
shall include those vertebrate species belonging to the classes Agnatha
(jawless fishes), Chondrichthyes (cartilaginous fishes), and
Osteichthyes (bony fishes).

3.5.2 OBJECTIVES

1. To provide data for surface water quality standards evaluation
and the National Pollutant Discharge Elimination System (NPDES)
permit program;

2. to provide data to assess human health concerns with special regard
to fish consumption; and

3. to provide complementary data for assessing water quality impacts
to aquatic and semi-aquatic biota.

3.5.3 FIELD SAMPLING

The collection of fish samples and field data pertaining to the objec-
tives stated above are conducted in cooperation with the Massachusetts
Division of Fisheries and Wildlife (MDFW) . The MDFW supplies one full-
time biologist and equipment when necessary. Fish are collected under
guidelines included in a "Scientific ^Collecting Permit for Fish" issued
to the Division of Water Pollution Control by the Division of Fisheries
and Wildlife. This permit is renewed annually.

Physical Measurements

When assessing water quality impacts as stated in objective 3.5.2(3)
data concerning stream reach length, width, and average depth are
recorded. Substrate characteristics are visually inspected and noted.
Water temperature is also recorded. Also under objective 3.5.2(3) all
fish are identified, weighed, and measured. Scales or spines are
sampled and used for aging. All fish are then released if they show
minimal stress. Under objectives 3.5.2 (1) and (2), only targeted
species of appropriate size are collected, identified, weighed, and
measured. These fish are brought back to the laboratory for processing.
In lakes and ponds, collection areas are marked on prepared maps, and
amount of effort (time) is recorded. When electrof ishing is performed
conductivity is recorded along with voltage used and relative success.

Gill Netting

Gill nets are entanglement gear best described as vertical walls of
netting. The typical net used by this Division is of an experimental
design. The nets are 38 meters in length and two meters in depth
stretched. They usually include a 1.27 cm polypropylene float line and
a 23 kg lead line. The net itself is composed of five 7.6 meter
monofilament panels. Mesh sizes are: 2.54 cm; 3.175 cm; 3.81 cm; 4.445
cm; and 5.08 cm.

68

FISH

Gill nets are set overnight for approximately 16-20 hours. The Division
is experimenting with two-hour sets to minimize the number of unwanted
fish collected. Nets are usually set in at least 2.5 m of water and
are marked by a buoy on each end. An additional buoy is attached near
the center of the net in water less than 3.0 m in depth to warn boaters
and/or fishermen of the obstruction.

- Electrof ishing

Electrof ishing using alternating current (a.c.) or direct current (d.c.)
is conducted in streams and in shallow water habitats in lakes, ponds
and impoundments. In lotic environments sampling begins and continues
until a satisfactory sample is attained at the lower end of a reach of
approximately 50-200 meters in length. Electrically-activated elec-
trodes are swept together along and under stream banks and around rocks,
logs and other obstructions. Stunned fish are collected with a dip-net
and placed in a tub of stream-water for later processing. Fish sampling
in lakes, impoundments and deep rivers is performed using a boat driven
slowly forward through shallow areas. In both types of habitats, an
estimate of the fish species and numbers missed is noted.

Trapping

Wooden cylindrical catfish traps are used to collect catfish and
bullheads (Ictaluridae) . These are baited, set in suitable locations,
and periodically checked. The trap has an opening on one end with a
cone-shaped entrance. The fish enter through the cone and cannot find
the entrance once in the box end of the trap.

Processing

Fish collected from each station are identified, weighed, measured, and
labeled, accordingly. Selected specimens are placed in plastic bags
and stored on ice in a cooler. Scales from representative fish are
collected and placed in "scale envelopes" for further analysis.

3.5.4 LABORATORY ANALYSIS

Processing

Fish collected for objectives 3.5.2 (1) and (2) are used for bioaccumu-
lation data analysis which is incorporated into public health determi-
nations or National Pollutant Discharge Elimination System permit
reviews. Each fish is weighed whole with entrails intact. Length is
measured from the tip of snout with mouth closed to the longest part of
the caudal fin slightly compressed. This is expressed as total length.

Each fish is rinsed with deionized water and filleted. A clean, sharp
fillet knife is run along each side of the backbone and then just to the
outside of the rib cage. This removes a boneless fillet from each side
of the fish. The fillet is then placed, skin down, on the filleting
board. A knife is used to separate the flesh from the skin. The skin
is discarded. One fillet, depending on the study, is either wrapped

69

individually, or composited with fillets from other fish of the same
species and size. The opposite fillet is wrapped individually, tagged
with a three or four letter code and number, and archived for future use.
Samples for metals analysis are wrapped in plastic (e.g. Saran) wrap.
Samples to be tested for PCB's are wrapped in household grade aluminum
foil. Fillets to be analyzed for dioxin are wrapped in aluminum foil
which has been rinsed with methanol and methylene chloride. The fillet-
ing board and knife are rinsed thoroughly after each fish is filleted.
Processed fish are kept frozen until they are transported to the analyt-
ical laboratory for analysis.

Fish are analyzed for metals and/or organics depending on the individual
study being performed. All results are reported as mg/kg. Quality
control and assurance data are recorded with each run of samples by the
analytical laboratory.

Aging

All fish collected are aged by use of scales or spines. Scales are
taken from various areas of a fish depending on the species being
sampled. Scales are dried in scale envelopes. The impressions are
made on butyrate slides, with a scale press. The impressions can then
be read off a scale reader or microfilm reader. Pectoral spines are
collected from Ictalurids. These spines are dried and cleaned of
excess skins and flesh. They are soaked in Axion detergent, which helps
loosen the skin and flesh which results in easier removal. Spines are
cross-sectioned at the basal recess on a low speed diamond bladed saw.
Cross-sections of .10-. 20 mm. can then be read through a compound micro-
scope. Ages are expressed as years+s for example 1+, 2+, 3+.

3.5.5 DATA MANAGEMENT

Reporting of Results

In most cases involving objectives 3.5.2 (1) and (2) results are put
into tabular form and a technical memorandum is written detailing the
nature of the study, methods used, and any applicable recommendations.
The memorandum is distributed to interested parties including the
Massachusetts Department of Public Health and the DEQE Office of
Research and Standards.

Computer Files

All fish data are entered into one of 4 DBase files. The files include
station identification information (STAID), a record of samples
(SAMPREC), the results of analyses for metals (FISHMET), and the results
for organics (FISHORG). These files are linked in such a manner that
data can be retrieved by species, waterbody, analyses type, concentra-
tion of contaminant, year, size, and other metrics. Data from these
files are the beginning of a statewide data base.

70

FISH

3.5.6 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories

| { state vehicle, clipboard

|~~f electrof ishing boat

| [ boat motor, gas can (and line)

f I generator and gas can

j [ generator tote barge

| [ anchor, rope

[ | life jackets, seat pads

j j fire extinguisher

| [ boat lights

Collecting and Sampling Gear
[~~] backpack electrof ishing gear

i J pocket thermometer

| | tape measure

[__} range finder

I [ plastic bucket, rope

(_J plastic bags (and ties)

j | glass and/or plastic vials

|"~~j glass and/or plastic jars, bottles

; i formalin

j | dip-nets

! [ gill nets

I [ fish measuring board

[ [ pan balance

Field Apparel

j i | rain gear (jacket, pants, hat)
1 [ hip boots and/or chest waders
J rubber gloves

Miscellaneous Items

i I USGS topographic maps

j [ clipboard

! j field data sheets, maps

j j length-weight, length-frequency forms

j ] tags and labels (with elastics or

string)

|~| pencils, pens

I j field identification manuals, keys

{ [ dissecting kit, hand lens

| [ aluminum foil and plastic wrap

' [ camera, film

[ [ first-aid kit

| | field glasses

\~ ~| insect repellent

H] tool kit

} [ cooler(s) , ice

j [ paper towels

Pj flashlights

I j ear protectors

71

3.5.7 DATA RECORD SHEETS AND FRESHWATER AND ANADROMOUS FISHES

CODING LIST

72

^

FISH

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

EXAMPLE OF SCALE (ENVELOPE)

WATERS

TOWN

TAG NO.

SP.

NO.

TL.

IN.

SL.

MM.

WGT.

SEX

M

G

STOM.

D

Mass. F. & W.

Fish Scale Record

!

73

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

FISH LENGTH - WEIGHT DATA SHEET

Collection Method:

Collector:

Date/Time:

Weather:

Water:

Station:

Species

Length

Weight

Species

Length

Weight

Species

Length

Weight

'•

•

-

74

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

FISH LF.NCTII - FREQUENCY DISTRIBUTION SIIEEr

FISH

Co 1 L jc t ion Mb tl iod :

Collector:

Date/Tine:

Weather:

Water:

SLation:

Milliliters

Tally

Milliliters

Tally

JO - 39

450 - 459

40 - 49

460 - 469

50 - 59

470 - 479

GO - 69

480 - 489

70 - 79

490 - 499

80 - 89

500 - 509

90 - 99

510 - 519

100 - 109

520 - 529

110 - 119

530 - 539

120 - 129

540 - 549

1.10 - 1J9

550 - 559

140 - 149

560 - 569

150 - 159

570 - 579

160 - 169

580 - 589

170 - 179

590 - 599

180 - 189

600 - 609

190 - 199

610 - 619

200 - 209

620 - 629

210 - 219

630 - 639

220 - 229

640 - 649

2J0 - 2J9

650 - 659

240 - 249

660 - 669

250 - 259

670 - 679

200 - 209
2 70 - 279

680 - 689

690 - 699

280 - 289
290 - 299
J00 - 309
3L0 - 319

700 - 709

710 - 719

720 - 729

730 - 739

320 - 329

330 - 339

740 - 749

750 - 759

340 - 349

760 - 769

350 - 359

770 - 779

360 - 369

780 - 789

370 - 379

790 - 799

380 - 389

800 - 809

390 - 399

810 - 819

400 - 409

820 - 829

410 - 419

830 - 839

420 - 429

840 - 849

430 - 439

850 - 859

440 - 449

860 - 869

Total Niwiljor

Total Weight
(Kg)

75

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79

3.5.8 REFERENCES

1. Bagenal, T. ed. 1978. Methods for Assessment of Fish Production in
Fresh Waters. IBP Handbook No. 3. Blackwell Scientific Publications,
Oxford, England. xvi + 365 p.

2. Clayton, G. , C. Cole, and S. Murawski. 1978. Common Marine Fishes of
Coastal Massachusetts. Massachusetts Cooperative Extension Service,
Amherst, x + 231 p.

3. Eddy, S., and J.C. Underhill. 1978. How to Know the Freshwater Fishes.
W.C. Brown Company, Publishers. Dubuque, Iowa. viii + 215 p.

4. Halliwell, D.B. 1981. A List of Freshwater Fishes of Massachusetts.
Massachusetts Division of Fisheries and Wildlife, Westborough. 12 p.

5. Lagler, K.F. 1971. Freshwater Fishery Biology. Wm. C. Brown Company,
Publishers. Dubuque, Iowa. xii + 421 p.

6. Mugford, P.S. 1969. Illustrated Manual of Massachusetts Freshwater
Fish. Massachusetts Division of Fisheries and Wildlife, Westborough.
v + 127 p.

7. Nielson, L.A. and D.C. Johnson, (eds.). 1983. Fisheries Techniques.
American Fisheries Society, Bethesda, Maryland. xvi + 468 p.

8. Robins, C.R., (ed). 1980. A List of Common and Scientific Names of
Fishes from the United States and Canada. Special Publication No. 12.
American Fisheries Society, Bethesda, Maryland. 176 p.

9. Scott, W.B. and E.J. Crossman. 1973. Freshwater Fishes of Canada.
Bulletin No. 184. Fisheries Research Board of Canada, Ottawa, Ontario,
Canada. xx + 966 p.

10. Smith, C.L. 1985. The Inland Fishes of New York State. The New York
State Department of Environmental Conservation, Albany. xii + 522 p.

80

MICROTOX'

SECTION

PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.6 Microtox™ Analysis

3.6.1 Definition

3.6.2 Objectives

3.6.3 Field Sampling
Qualitative
Quantitative

Sample Container Preparation
Sample Collection and Handling

3.6.4 Laboratory Analysis

Laboratory Equipment and Related Supplies

3.6.5 Quality Assurance

3.6.6 Field Equipment and Supply List

3.6.7 Interpretation and Reporting of Microtox™ Results
Test Description

Data Interpretations

Microtox™ Results Reporting Form

3.6.8 Microtox™ Sediment Toxicity Testing
Laboratory Equipment and Related Supplies

3.6.9 References

82
82
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88

81

3.6 MICROTOX™ ANALYSIS

3.6.1 DEFINITION: The Microtox™ toxicity analyzer uses a lyophilized (freeze-
dried) marine bioluminescent bacterium (Photobacterium phosphoreum)
which, upon reconstitution, emits a fairly constant level of light.
Upon exposure to a toxicant, the level of bioluminescence is diminished
in direct proportion to the toxicant concentration.

3.6.2 OBJECTIVES:

1. To assess the effectiveness of the Commonwealth's municipally-owned
and industrial wastewater treatment plants in eliminating or pre-
venting aquatic toxicity;

2. to selectively screen water and sediment samples prior to per-
forming more expensive and time-consuming conventional toxicity
tests ;

3. to determine the toxicity of known toxicants using laboratory-
prepared solutions of known concentrations; and

4. to compare Microtox™ test results with results from other toxicity
tests.

3.6.3 FIELD SAMPLING

Qualitative

Because the Microtox"1 system is designed as a quantitative test,
qualitative results cannot be determined.

Quantitative

Based on the objectives of the study and an understanding of the short-
and long-terra operations and schedules of the discharger, either grab •
or composite-type samples are collected for testing. If the suspected
toxicity of the source is variable, grab samples collected during peaks
of toxicity provide a measure of maximum impact. The compositing
technique has an averaging effect, which tends to dilute toxicity peaks,
and may provide misleading results when testing for acute toxicity.
Composite samples, therefore, are more appropriate for chronic toxicity
tests where peak toxicity of short duration is of less concern.

Sample Container Preparation

The 450 ml borosilicate-type glass containers are prepared according to
the methods described in the United States Environmental Protection
Agency's Handbook for Sampling and Sample Preservation of Water and
Wastewaters (See: "References"), unless the containers are previously
unused.

82

MICROTOX'

Sample Collection and Handling

Sample containers are rinsed once with sample water prior to collection.
An effort is made to fill the sample container to near capacity, with
little or no air space. After the container is filled with the sample,
a pH reading is taken by the collector with an Orion Model 201 field pH
meter. Plastic wrap is placed under the container cap and the sample is
put on ice for transport back to the Microtox™ laboratory.

Upon arrival at the Microtox™ laboratory, the sample is either (1)
tested or (2) refrigerated until the following day and then tested. The
maximum holding time for a sample after collection is 24 hours.

3.6.4 LABORATORY ANALYSIS

The basic procedure for the Microtox™ system employs duplicates of a
non-toxic control and- four serial dilutions of the sample. The mean
response of the duplicate control is used to normalize the duplicate
responses of the four test concentrations of sample when the test
results are reduced. Detailed operating procedures for using the
Microtox™ Analyzer are found in the Microtox System Operating Manual
(see: "References").

Laboratory Equipment and Related Supplies

1. Beckman Microtox™ model 2055 toxicity analyzer

2. strip chart recorder, chart paper

3. Microtox™ reagent ( lyophilized)

4. Microtox™ reagent diluent

5. Microtox™ reconstruction solution

6. Microtox™ osmotic adjusting solution

7. cuvettes, glass, disposable [11.75 mm x 50 mm in size]

8. recorder pen, black

9. Eppendorf 10 ul pipet, micropipette tips 1-100 ul

10. Eppendorf 500 ul pipet, micropipette tips 101-1000 yl

11. parafilm, kimwipes

12. disposable gloves

3.6.5 QUALITY ASSURANCE

Every tenth sample is tested in duplicate to check consistency and
reproducability of results.

83

3.6.6 FIELD EQUIPMENT AND SUPPLY LIST

Collecting and Sampling Gear

'■~\ 450 ml borosilicate type glass containers with caps

^J Orion model 201 field pH meter

I rubber gloves

Miscellaneous Items
i_J tags, labels, elastics
_J pencils, pens
[ plastic wrap

first aid kit

[ cooler, ice

84

MICROTOX'

3.6.7 INTERPRETATION AND REPORTING OF MICROTOX RESULTS
Test Description

Microtox™ is the trade name for a particular acute toxicity test. The test is
used as a toxics screening tool in addition to other, more traditional, methods
of analysis.

The Microtox™ analyzer uses freeze-dried luminescent bacteria as its test
organisms. When re-hydrated, these bacteria emit light. To test a water sample
for toxicity using Microtox™, an analyst prepares a series of dilutions of the
sample and adds re-hydrated bacteria to these. The light intensity of each
sample dilution is measured at preselected time intervals over a 30-minute
period and compared with that of a control (bacteria only). It is assumed that
changes in light intensity are due to toxicant interference with the biochemical
reaction that produces light. Toxicity is then measured as the percent decrease
in light intensity of each of the sample dilutions compared with that of the
control.

Data Interpretations

The most commonly used result from these tests is the 30-minute EC5Q. This is
defined as the sample Concentration causing a 50% reduction in the measured
Effect (light production) over a 30-minute time period. The relationship of the
EC5Q to toxicity is an inverse one; i.e., the lower the EC50, the greater the
toxicity of the sample.

A useful conversion of the ECcq is the Toxic Unit. This is simply the inverse
of the EC5Q multiplied by a factor of 100:

Toxic Units = 100

EC50 (%)

Toxic Units approximate the amount of dilution a sample must undergo so as not
to induce a toxic response in the test organisms (the Microtox™ bacteria).
As Toxic Units increase, so does the relative toxic strength of a sample. The
relationship of EC5q's, Toxic Units, and toxicity are demonstrated below:

EC50 (>0 Toxic Units Toxicity

0.5 200 High

1.0 100

10.0 10

100.0 1 Low

Samples not toxic enough to produce a full 50% decrease in light over the time
allotted for the test may still be toxic enough to produce a response in the
test. The EC20 and E^10 (3ample concentrations causing a 20% and 10% reduction
in light intensity respectively) are reported in order to give the regulator an
idea of incipient toxicity - sample dilutions which induce a small, but
measurable response in the test.

85

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

MICROTOX™ RESULTS REPORTING FORM

SAMPLES TESTED

LOG #

SITE

SAMPLE TYPE

DATE COLLECTED

DATE TESTED

COLLECTOR

FIELD pH

LAB pH

HARDNESS

SPEC. COND.

LOG #

5 MIN.

MICROTOX™ RESULTS

15 MIN.

30 MIN.

EC10

EC20

EC50

TOXIC
UNITS
(T.U.)

NOTE: RESULTS GIVEN AS % VOLUME OF SAMPLE

86

MICROTOX'

Results of the Microtox™ test are also reported for three different periods
of exposure: 5-minute, 15-minute, and 30-minute. A decrease in the EC5Q over
time (increase in Toxic Units) usually indicates the presence of persistent
toxicants (e.g., metals) in the sample. An increase in the EC50 over time
(decrease in Toxic Units) suggests that non-persistent toxics (e.g., volatile,
biodegradables , photo or hydrolyzible material) are present at time of sampling,

3.6.8 MICROTOX" SEDIMENT TOXICITY TESTING

The Microtox™ bioassay can also be used to determine the toxicity of the water
soluble fraction (WSF) of sediment samples. Detailed sample preparation proce-
dures are found in the U.S. Environmental Protection Agency's draft Permit
Guidance Manual on Hazardous Waste Low Treatment Demonstrations (See:
"References") .

Laboratory Equipment and Related Supplies

1. Eberbach shaker table - small tabletop model with carrying tray

2. IEC high speed centrifuge model HN

3. Mettler balance

4. Dessicator

5. Drying oven

6. Evaporating dishes

7. Fleaker beakers

8. Centrifuge tubes

9. Graduated cylinders
10. Tongs

87

3.6.9 REFERENCES

1. Beckman, Inc. 1980. Microtox™ Model 2055 Toxicity Analyzer System.
Bulletin 6984. Beckman Instruments, Inc., Carlsbad, CA. 8 p.

2. Beckman, Inc. 1982a. Microtox™ Application Notes No. M304: Toxicity
Testing of Complex Effluents. Beckman Instruments, Inc., Carlsbad, CA.
2 p.

3. Beckman, Inc. 1982b. Microtox™ System Operating Manual. Beckman
Instruments, Inc., Carlsbad, CA. 59 p.

4. Fitzgerald, F.X. 1985. Unpublished Microtox™ Program Notebook.
Massachusetts Division of Water Pollution Control, Technical Services
Branch, Westborough, MA. (Unpaginated) .

5. Peltier, W.H. and C.I. Weber. 1985. Methods for Measuring the Acute
Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-85-
013. United States Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio. xvi + 216 p.

6. Sheehan, K.C., K.E. Sellers, and N.M. Ram. 1984. Establishment of a
Microtox™ Laboratory and Presentation of Several Case Studies Using
Microtox™ Data. Env. Eng. Report No. 77-83-8. University of
Massachusetts, Department of Civil Engineering, Amherst, MA. viii + 76 p

7. United States Environmental Protection Agency. 1982. Handbook for
Sampling and Sample Preservation of Water and Wastewater. EPA-600/4-28-
029. U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio. xii + 402 p.

8. United States Environmental Protection Agency. 1984. Permit Guidance
Manual on Hazardous Waste Land Treatment Demonstrations. Draft EPA-530-
SW-84-015. U.S. Environmental Protection Agency, Office of Solid Waste
and Emergency Response, Washington, D.C. xiii + 123 p.

88

CHLOROPHYLL

SECTION

PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.7 Chlorophyll Analysis

3.7.1 Definition

3.7.2 Equipment Needs

3.7.3 Log-In Procedure

3.7.4 Sample Preparation

3.7.5 Analytical Procedure

Calculation of Chlorophyll Concentrations

3.7.6 Instrument Calibration

3.7.7 References

90
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95

89

3.7

CHLOROPHYLL ANALYSIS

3.7.1 DEFINITION: Chlorophyll is a pigment found in plants that allows the
organism to use radiant energy for converting carbon dioxide into
organic compounds in a process called photosynthesis. Several types of
chlorophylls exist and these and other pigments are used to characterize
algae. One type, chlorophyll a, is measured for it is found in all
algae. A knowledge of chlorophyll a concentrations provides qualitative
and quantitative estimations of phytoplanktonic and periphytic biomasses
for comparative assessments of geographical, spacial and temporal
variations.

3.7.2 EQUIPMENT NEEDS

1. Fluorometer - either Turner 111 or the Turner Design 10-005-R
field fluorometer is used. They must be equipped with blue lamp
F4T5.

Corning filter - 5-60-excitation
Corning filter - 2-64-emission
Photomultiplier

2. Tissue grinder and tube - Thomas Tissue Grinder

3. Side arm vacuum flask and pump

4. Millipore filter holder

5. Glass fiber filter: Reeve angel, grade 934H, 2.1 cm

6. Centrifuge (Fisher Scientific Safety Centrifuge)

7. 15 ml graduated conical end centrifuge tubes with rubber stoppers

8. 90% aqueous acetone

9. IN HCL

10. Saturated magnesium solution in distilled water

11. Test tube racks

12. Borosilicate cuvettes -

Turner 111 - 3" cuvettes
Turner Design - 8" cuvettes

13. Aluminum foil

14. Test tube brushes - conical end

15. Parafilm

90

CHLOROPHYLL

3.7.3 LOG-IN PROCEDURE

As samples are received they are logged in and assigned a number. The
samples can be frozen for further analysis, or the filter ground up for
analysis the following day.

3.7.4 SAMPLE PREPARATION

Samples are generally processed as soon as they come into the labora-
tory, unless there are extenuating circumstances, such as faulty
equipment and/or time constraints. Samples not to be analyzed within
24 hours are frozen for future analysis.

The procedure for freezing samples follows:

1) Label a 2-inch Whatman petri dish with the sample number using an
indelible pen.

2) Using tweezers, take a 2.1 cm Reeve Angel, grade 934AH, glass fiber
filter and place it on the Millipore filtering flask screen. Do
not touch the filter. Attach the glass tube to the filter flask
with the metal clamp.

3) Shake the sample well.

' 4) Measure out 50 mis of sample or less. If an amount other than 50
mis is used it should be recorded in the chlorophyll data book.

5) Pour the measured sample into the filter tube and turn on the
vacuum. The sample should pass quickly through the glass fiber
filter; therefore more of the sample should be added. If the
sample is not filtering through - either because too much sediment
is present or the algal concentration is too high - then less than
50 mis can be filtered. A notation is made in the chlorophyll
data book which lists the amount that was filtered.

6) Unclamp the filter holder and with tweezers transfer the filter to
the previously marked petri dish.

7) Cover the petri dish and wrap it in aluminum foil to keep out the
light. The petri dish with the glass fiber filter is then stored
in the freezer.

8) Return the sample bottle to the refrigerator if algal counts or
identifications are requested.

9) Rinse the graduated cylinder and filter holder in distilled water.

91

3.7.5 ANALYTICAL PROCEDURE

1) Follow steps 2-6 under "Sample Preparation."

2) Filter 50 ml (or less if necessary) of sample through a glass fiber
filter under vacuum.

3) Push the filter to the bottom of tissue grinding tube.

4) Add about 3 ml of 90% acetone and 0.2 ml of the MgC03 solution.

5) Grind contents for 3 minutes.

6) The contents of the grinding tube are carefully washed into a 15 ml
graduated centrifuge tube.

7) Bring the sample volume to 10 ml with 90% acetone.

8) Test tubes are wrapped with aluminum foil and stored in the
refrigerator for 24 hours.

9) Test tubes are taken out of the refrigerator and put into the
centrifuge.

10) Test tubes are then centrifuged for 20 minutes and the supernatant
decanted immediately into stoppered test tubes.

11) Tubes are allowed to come to room temperature. The temperature is
recorded and the samples are poured into a cuvette (3" for Turner
111 and 8" for Turner Design).

12) The Turner 111 requires a warm-up period of at least one-half hour,
while the Turner Design 10-005-R does not require a warm-up period.

13) With Turner 111, use a blank of 90% aqueous solution of acetone to
zero the instrument. Open the front door of the fluorometer and
put in the cuvette containing the 90% acetone and close the door.
Press the start switch. The dial should move back to 0; adjust-
ments can be made with the calibration knob. This process should
be repeated as often as necessary, i.e., if the blank is not
staying on zero; but no alteration should be made until a series
of samples is completed.

14) The Turner Design must also be zeroed to an acetone blank. The
sample holder is located at the top of the Turner Design field
fluorometer and should be recovered with the black cap after the
sample is put in it.

15) Readings for both the Turner 111 and the Turner Design should be
within 20-80% of the scale. This can be achieved by either
reducing or increasing the opening to the lamp by moving the knob
on the right front of the Turner 111 fluorometer. The sensitivity
levels are lx, 3x, lOx, and 30x. The sensitivity level must be
recorded in the chlorophyll data book in addition to whether the
high intensity or regular door was used. After the first reading,
2 drops of 2N HCl is added to the cuvette. A piece of parafilm is
used to cover the cuvette which is then inverted four times to mix
the sample thoroughly. The sample is re-read and the new value
recorded.

92

CHLOROPHYLL

16) The procedure for the Turner Design field fluorometer is basically
the same as for the Turner 111. The sample is put into the cuvette
holder and the manual switch used to go from one sensitivity level
to the next without opening the door. A reading of between 20-80%
is still required for accuracy. Readings are taken before and
after acid is added to the sample. The level of sensitivity (lx,
3x, 6x, lOx, 31. 6x) must be recorded in the chlorophyll data book,
as well as whether the levels were set at 1 or 100.

Calculation of Chlorophyll Concentrations

Chlorophyll concentrations are determined by using the following
formulas :

chlorophyll (ug/1) = Fs ££_ (Rb-RA)

rs-1

pheophytin (ug/1) = Fs ££_ (rsRa-Rb)

rs-1

where,

Fs = conversion factor for sensitivity level "s"

rs = before and after acidification ratio of sensitivity level "s"

Rb = fluorometer reading before acidification

Ra = fluorometer reading after acidification

A computer program is used to calculate the chlorophyll concentrations
for samples run on the Turner Design fluorometer. This program requires
the investigator to type in the sensitivity level and the difference
between the before and after acidification values.

During the summer of 1986 personnel of the Technical Services Branch
(TSB) conducted a laboratory experiment with a Turner Design Fluorometer
in order to determine the effect of pheophytin b on freshwater chloro-
phyll a readings. Pheophytin b_ is the degradation product of
chlorophyll b_ which is the primary pigment of green algae. The Turner
Design instrument measures the fluorescence of chlorophyll a as well as
that of pheophytin a and b. Chlorophyll b is not read at the same
frequency as chlorophyll a. The emission filter used at the TSB
(Corning C/S 2-64) partially rejects pheophytin b^ (See: "References'1
- Turner Designs, 1981). It was found and recorded in various
unpublished memoranda (See "References") that unless a sample had ele-
vated counts of green algae the readings obtained prior to acidification
and 90 seconds thereafter would give a reliable estimate of the concen-
tration of chlorophyll a in an algal sample. In cases with elevated
counts of green algae an annotation should be made alongside the
chlorophyll a concentration stating that the concentration may reflect
the presence of chlorophyll b and is probably lower than as recorded.
As a result of this investigation, the TSB now presents chlorophyll data
as chlorophyll a in rag/m->.

93

3.7.6 INSTRUMENT CALIBRATION

Fluorometers are calibrated using chlorophyll samples provided by the
United States Environmental Protection Agency. Calibrations are
performed at the start of every field season and redone if any changes
are made to the fluorometer such as changing the light bulb.

Samples for chlorophyll analysis are periodically split with another
laboratory or run on two separate fluorometers.

94

CHLOROPHYLL

3.7.7 REFERENCES

1. • Beskenis, J.L. 1984. CHLA Program (Unpublished). Massachusetts

Division of Water Pollution Control, Westborough. (Unpaginated) .

2. Beskenis, J.L. 1985. CHLORA Program (Unpublished). Massachusetts
Division of Water Pollution Control, Westborough. (Unpaginated).

3. Beskenis, J.L. 1986. CHL086 Program (Unpublished). Massachusetts
Division of Water Pollution Control, Westborough. (Unpaginated).

4. Greenberg, A.E., R.R. Trussell, L.S. Clesceri, and M.H. Franson, (eds.).
1985. Standard Methods for the Examination of Water and Wastewater.
American Public Health Association, Washington, D.C. xlix + 1268 p.

5. Kimball, W.A. 1976. Procedure for Chlorophyll Analysis (Unpublished).
Massachusetts Division of Water Pollution Control, Westborough.
(Unpaginated) .

6. Ryan, K.M. 1986. Preliminary Study on Chlorophyll Analysis (Unpublished
memorandum dated July 7, 1986.) Massachusetts Division of Water
Pollution Control, Westborough. (Unpaginated).

7. Ryan, K.M. 1986. Study on Chlorophyll Analysis (Unpublished memorandum
dated August 25, 1986). Massachusetts Division of Water Pollution
Control, Westborough. (Unpaginated).

8. Turner Designs. 1976. Operating and Service Manual (Model 10 Series
f luorometers) . Mountain View, CA. ii + 35 p.

9. Turner Designs. 1981. Fluorometric Facts - Chlorophyll and Pheophytin
(Bulletin 101). Mountain View, CA. 12 p.

10. Vollenweider , R.A. , (ed.). 1974. A Manual on Methods for Measuring
Primary Production in Aquatic Environments. IBP Handbook No. 12.
Blackwell Scientific Publications, Oxford, England. xviii + 225 p.

11. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA-670/4-73-001 .
United States Environmental Protection Agency, National Environmental
Research Center, Cincinnati, Ohio. xii + 146 p. + appendices.

95

4.0 QUALITY ASSURANCE

96

QUALITY ASSURANCE

SECTION

4.0 QUALITY ASSURANCE

4.1 Purpose and Scope

4.2 Intralaboratory Quality Assurance

4.3 Interlaboratory Quality Assurance

4.4 References

PAGE

98

98

98

99
100

97

4.0 QUALITY ASSURANCE

4.1 PURPOSE AND SCOPE

A quality assurance program has been put in place to validate both the
reliability of field and laboratory techniques and the integrity of the
biotnonitoring data. An essential element of this program is the
development of standardized field and laboratory methodologies as out-
lined in this manual of operating procedures. Standard methods allow
for the determination of the accuracy, precision, and variability of
biomonitoring data.

Although details pertaining to the quality assurance program have
already been presented for individual biomonitoring program elements,
major components of the program that are applicable to most biomonitor-
ing activities are summarized in this section.

4.2 INTRALABORATORY QUALITY ASSURANCE

1) A staff of adequately trained aquatic biologists is maintained;
each with knowledge of the taxonomy and pollution ecology of one
or more freshwater communities . These include bacteria, algae,
macrophyton, aquatic macroinvertebrates , and fish.

2) Collecting gear such as nets, sieves, and grab samplers are
inspected and maintained frequently.

3) Field and laboratory equipment such as pH and dissolved oxygen
meters, microscopes, and fluorometers are maintained and calibrated
on a routine basis.

4) Field studies are carefully planned in advance to insure that
appropriate sites are sampled and that the proper number of samples
are obtained to meet survey goals and objectives.

5) All samples are clearly labeled at the time of collection, recorded
in hard-bound log books, and tracked in a step-wise fashion
throughout their processing in the laboratory.

6) A reference library is maintained which includes up-to-date identi-
fication manuals and keys and both benchmark and recent literature
on all aspects of water pollution and its impact on aquatic life.

7) A reference specimen collection is maintained for confirming the
proper identification of aquatic invertebrates. Similar
collections for other communities (e.g., fish) are under develop-
ment. In addition, many reference specimens and other organisms
of interest are photographed and added to an extensive collection
of slides to be used as taxonomic aids and for training purposes.

98

QUALITY ASSURANCE

8) Aquatic macroinvertebrate, algae, chlorophyll, and Microtox™ data
are input to computerized data storage and retrieval systems
insofar as is allowed by time and personnel constraints. All data
sets are carefully proofread and edited during this process. A
similar system is proposed for the storage of data generated by the
fish sampling program.

9) All reporting elements receive peer and/or supervisory review and
numerical analyses are checked for mathematical errors.

4.3 INTERLABORATORY QUALITY ASSURANCE

1) Reference samples containing known chlorophyll a concentrations,
predetermined phytoplankton counts, or known invertebrate taxa are
routinely provided to the biomonitoring staff by the United States
Environmental Protection Agency (U.S. EPA) for instrumentation
calibration and evaluation of laboratory performance.

2) Occasionally biological surveys are conducted simultaneously with
the USEPA or other state agencies to compare field and laboratory
methods and to determine interlaboratory variability of results.

3) Specimens that present particular problems with their identifica-
tion are often sent to expert taxonomists for confirmation. A
separate log book is used to record -the date and • to whom specimens
are sent, and, ultimately, the date and details pertaining to the
taxonomists1 responses.

99

4.4 REFERENCES

1. Crim, R.L., (ed.). 1975. Model State Water Monitoring Program.
EPA-440/9-74-002. United States Environmental Protection Agency, Office
of Water and Hazardous Materials, Washington, D.C. viii + 58 p.

2. Greenberg, A.E., R.R. Trussell, L.S. Clesceri, and M.H. Franson, (eds).
Standard Methods for the Examination of Water and Wastewater. American
Public Health Association, Washington. xlix + 1268 p.

3. United States Environmental Protection Agency. 1972. Handbook for
Analytical Quality Control in Water and Wastewater Laboratories. Analytical
Quality Control Laboratory, Cincinnati, Ohio, xii + 99 p.

4. U.S. Environmental Protection Agency. 1982. Handbook for Sampling and
Sample Preservation of Water and Wastewater. EPA-600/4-82-029
Environmental Monitoring and Support Laboratory, Cincinnati. xii +
402 p.

5. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA-670/4-73-001 .
U.S. EPA, National Environmental Research Center, Cincinnati. xii +

146 p. + appendices..

100

5.0 GENERAL BIOLOGICAL FIELD AND LABORATORY REFERENCES

!

101

5.0 REFERENCES

1. Anderson, R.M. 1965. Methods of Collecting and Preserving Vertebrate
Animals. National Museum of Canada Bulletin No. 69. Supply and Services
Canada, Ottawa, viii + 199 p.

2. Bagenal, T., (ed.). 1978. Methods for Assessment of Fish Production in
Fresh Waters. IBP Handbook No. 3. Blackwell Scientific Publications,
Oxford, England, xvi + 365 p.

3. Bordner, R. and J. Winter, (eds.). 1978. Microbiological Methods for
Monitoring the Environment. EPA-600/8-78-017. United States Environmental
Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio. xvi + 338 p.

4. Cairns, J., Jr. and K.L. Dickson, (eds.). 1973. Biological Methods for
the Assessment of Water Quality - A Symposium Presented at the Seventy-
fifth Annual Meeting of American Society for Testing and Materials. ASTM
Special Technical Publication 528. ASTM, Philadelphia, Pennsylvania,
viii + 256 p.

5. Cairns, J., Jr., (ed.). 1982. Artificial Substrates. Ann Arbor Science
Publishers, Inc., Ann Arbor, Michigan. xiv + 279 p.

6. Crim, R.L., (ed.). 1975. Model State Water Monitoring Program. EPA-440/
9-74-002. U.S. EPA, Office of Water and Hazardous Materials, Washington,
D.C. viii + 58 p.

7. Edmondson, W.T. and G.G. Winberg, (eds.). 1971. A Manual on Methods for
the Assessment of Secondary Productivity in Fresh Waters. IBP Handbook
No. 17. Blackwell Scientific Publications, Oxford. xxiv + 358 p.

8. Forsberg, C. 1959. Quantitative Sampling of Subaquatic Vegetation. Oikos
10(2):233-240. ~

9. Gonor, J.J. and P.F. Kemp. 1978. Procedures for Quantitative Ecological
Assessments in Intertidal Environments. EPA-600/3-78-087. U.S. EPA,
Environmental Research Laboratory, Corvallis, Oregon. viii + 104 p.

10. Green, R.H. 1979. Sampling Design and Statistical Methods for Environ-
mental Biologists. John Wiley and Sons, New York. xiv + 257 p.

11. Greenberg, A.E., R.R. Trussell, L.S. Clesceri, and M.H. Franson, (eds.).
1985. Standard Methods for the Examination of Water and Wastewater.
American Public Health Association, Washington, D.C. xlix + 1268 p.

12. Holme, N.A. and A.D. Mclntyre, (eds.). 1971. Methods for the Study of
Marine Benthos. IBP Handbook No. 16. Blackwell Scientific Publications,
Oxford. xii + 334 p.

13. Keup, L.E., W.M. Ingram, and K.M. MacKenthun, (eds.). 1967. Biology of
Water Pollution - A Collection of Selected Papers on Stream Pollution,
Waste Water, and Water Treatment. United States Department of the
Interior, Federal Water Pollution Control Administration, Cincinnati,

ii + 290 p.

102

14. Kittrell, F.W. 1969. A Practical Guide to Water Quality Studies of
Streams. U.S. Department of the Interior, Federal Water Pollution Control
Administration, Washington, D.C. xii + 135 p.

15. Knudsen, J.W. 1966. Collecting and Preserving Plants and Animals. Harper
and Row, Publishers, New York. x + 320 p.

16. Lind, O.T. 1974. Handbook of Common Methods in Limnology. C.V. Mosby
Company, St. Louis, Missouri. viii + 154 p.

17. MacKenthun, K.M. 1969. The Practice of Water Pollution Biology. U.S.
Department of the Interior, Federal Water Pollution Control Administration,
Washington, D.C. xii + 281 p.

18. McCauley, V.J.E. 1975. Two New Quantitative Samplers for Aquatic
Phytomacrofauna. Hydrobiologia 47(l):81-89.

19. Schwoerbel, J. 1970. Methods of Hydrobiology (Freshwater Biology).
Pergamon Press, Inc., Elmsford, New York. x + 200 p.

20. Slack, K.V., R.C. Averett, P.E. Greeson, and R.G. Lipscomb. 1973. Methods
for Collection and Analysis of Aquatic Biological and Microbiological
Samples. Techniques of Water-Resources Investigations of the United
States Geological Survey, Chapter A4, Book 5 (Laboratory Analysis).
Superintendent of Documents, Washington, D.C. vi + 165 p.

21.. Sorokin, Y..I. and H. Kadota, (eds.). 1972. Techniques for the Assessment
of Microbial Production and Decomposition in Fresh Waters. IBP Handbook
No. 23. Blackwell Scientific Publications, Oxford. xvi + 112 p.

22. Southwood, T.R.E. 1978. Ecological Methods - with Particular Reference
to the Study of Insect Populations. Halsted Press, New York. xxiv +
524 p.

23. Swartz, R.C. 1978. Techniques for Sampling and Analyzing the Marine
Macrobenthos. EPA-600/3-78-030. U.S. EPA, Environmental Research
Laboratory, Corvallis. viii + 27 p.

24. United States Environmental Protection Agency. 1978. Environmental
Assessment Manual. Region I, Boston. 242 p.

25. U.S. Environmental Protection Agency. 1980. Microscopic Analysis of
Activated Sludge. EPA-430/ 1-80-007. National Training and Operational
Technology Center, Cincinnati. iv + 446 p.

26. U.S. Environmental Protection Agency. 1982. Handbook for

Sampling and Sample Preservation of Water and Wastewater. EPA-600/4-82-
029. Environmental Monitoring and Support Laboratory, Cincinnati,
xii + 402 p.

27. United States Geological Survey. 1972. Recommended Methods for Water
Data Acquisition. Office of Water Data Coordination, Washington, D.C.
iv + 394 p.

103

28. U.S. Geological Survey. 1977. National Handbook of Recommended Methods
for Water-Data Acquisition. Office of Water Data Coordination, Reston,
Virginia. i + 741 p.

29. Vollenweider , R.A. , (ed.). 1974. A Manual on Methods for Measuring
Primary Production in Aquatic Environments. IBP Handbook No. 12.
Blackwell Scientific Publications, Oxford, England. xviii + 225 p.

30. Weber, C.I., (ed.). 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. EPA-670/4-73-001 .
U.S. EPA, National Environmental Research Center, Cincinnati, xii +

146 p. + appendices.

31. Welch, P.S. 1948. Limnological Methods. McGraw-Hill Book Co., Inc.,
New York. xviii + 381 p.

32. Yevich, P.P. and C.A. Barszcz. 1977. Preparation of Aquatic Animals for
Histopathological Examination. U.S. EPA, Environmental Monitoring and
Support Laboratory, Cincinnati. ii + 20 p.

104