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Standard Operating Procedures

BIOMONITORING PROGRAM 1990

Massachusetts Department of Environmental Protection

DIVISION OF WATER POLLUTION CONTROL
Arleen O'Donnell, Acting Director

Publication # 16,326 - 147 pgs - 25 cps. 5/90 CR
Approved by: Ric Murphy, State Purchasing Agent

NOTICE OF AVAIIABILITY

LIMITED COPIES OF THIS REPORT ARE AVAILABLE AT NO COST BY WRITTEN REQUEST TO:

MASSACHUSETTS DEPARTMENT OF ENVIRONMENTAL PROTECTION

TECHNICAL SERVICES BRANCH

WESTVIEW BUILDING, LYMAN SCHOOL GROUNDS

WESTBOROUGH, MA 01581

Furthermore, at the time of first printing, eight (8) copies of each report
published by this office are submitted to the State Libary at the State House
in Boston; these copies are subsequently distributed as follows:

* On shelf; retained at the State Library (two copies) ;

* microfilmed; retained at the State Library;

* delivered to the Boston Public Library at Copley Square;

* delivered to the Worcester Public Library;

* delivered to the Springfield Public Library;

* delivered to the University Library at UMass, Amherst;

* delivered to the Library of Congress in Washington, D.C. ;

Moreover, this wide circulation is augmented by inter-library loans from the
above-listed libraries. For example, a resident of Winchendon can apply at
the local library for loan of the Worcester Public Library's copy of any
DWPC/TSB report.

A complete list of reports published since 1963 is updated annually and
printed in July. This report, entitled "Publications of the Technical
Services Branch, 1963- (current year)", is also available by writing to the
TSB office in Westborough.

BIOMONITORING PROGRAM

STANDARD OPERATING PROCEDURES

1990

Technical Services Branch
Massachusetts Division of Water Pollution Control
Department of Environmental Protection

Westborough

Executive Office of Environmental Affairs
John P. DeVillars, Secretary

Department of Environmental Protection
Daniel S. Greenbaum, Commissioner

Division of Water Pollution Control
Arleen O'Donnell, Acting Director

May 1990

TITLE

DATE

Biomonitoring Program Standard Operating Procedures
May 1, 1990

AUTHOR(S): Anonymous

REVIEWED BY

Arthur S. Johnson
Biomonitoring Program Manager

APPROVED BY:

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Alan N. CoopetfTnan , M.S., P.E.
Environmental Engineer VI

11

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 28

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS 33

3.1 Phytoplankton 34

3.2 Periphyton 48

3.3 Aquatic and Wetland Vegetation 57

3.4 Aquatic Macroinvertebrates 72

3.5 Fish 88

3.6 Caged Minnow Toxicity Assessments 101

3.7 Microtox™ Analysis 112

3.8 Chlorophyll Analysis 121

4.0 QUALITY ASSURANCE 128

5.0 LABORATORY AND SAFETY CONCERNS 133

6.0 GENERAL BIOLOGICAL FIELD AND LABORATORY REFERENCES 139

in

Digitized by the Internet Archive

in 2012 with funding from

Boston Library Consortium Member Libraries

http://archive.org/details/biomonitoringpro1990mass

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, macrophyton, 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
teratogens). 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

site.

5

2.1.5 DATA RECORD SHEETS

STREAM CLASSIFICATION

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MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

STREAM CLASSIFICATION FIELD DATA RECORD

RIVER:

DRAINAGE:

MUNICIPALITY

TOPOGRAPHIC MAP(S)

SITE:

COLLECTORCS)

DATE

TIME SAMPLING DURATION:

WEATHER/ATMOSPHERIC CONDITIONS:

AIR TEMPERATURE

WIND

Force -

Direction -

CLOUD CLASSIFICATION

Low

Middle

High

Percent Sky Coverage

PRECIPITATION (including

previous

day's)

DATA COLLECTIONS

PHYSICAL

WATER

HYDROLOGIC

BIOLOGICAL

OTHER OBSERVATIONS:

INSTRUMENT CALIBRATION

8

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

AQUATIC MACROINVERTEBRATE RAPID BIOASSESSMENT

SECTION PAGE

2.2 AQUATIC MACROINVERTEBRATE 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 Aquatic Macroinvertebrates and Assigned Tolerance Values 15

2.2.6 References 27

11

2.2 AQUATIC MACROINVERTEBRATE 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).

12

MACRO INVERTEBRATE RAPID BIOASSESSMENT

Field observations are 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 is difficult. Lacking any better information the values
are assigned as: 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 sen-
sitivity 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 less than 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 the individuals; oligochaete worms comprise less than
20% 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

AQUATIC MACRO INVERTEBRATE RAPID BIOASSESSMENT

2.2.5 AQUATIC MACRO INVERTEBRATES AND ASSIGNED TOLERANCE VALUES

TAXON NAME

TOLERANCE
VALUE

ANNELIDA

OLIGOCHAETA (AQUATIC EARTHWORMS)
LUMBRICULIDA
LUMBRICULIDAE

LUMBRICULUS SP.
HAPLOTAXIDA

ENCHYTRAEIDAE
NAIDIDAE
DERO SP

HAEMONAIS WALDVOGELI
NAIS SP.
NAIS BEHNINGI
NAIS COMMUNIS
NAIS PARDALIS
NAIS VARIABILIS
OPHIDONAIS SERPENTINA
SPECARIA SP.
TUBIFICIDAE

AULODRILUS SP.

IMMATURE W/O CAPILLIFORM SETAE
IMMATURE W/O CAPILLIFORM SETAE TYPE 1
IMMATURE W/O CAPILLIFORM SETAE TYPE 2
IMMATURE WITH CAPILLIFORM SETAE
LIMNODRILUS SP.
LIMNODRILUS HOFFMEISTERI
TUBIFEX SP.
TUBIFEX TUBIFEX
HIRUDINEA (LEECHES)
RHYNCHOBDELLIDA
GLOSSOPHONIIDAE

HELOBDELLA STAGNALIS
PHARYNGOBDELLIDA
ERPOBDELLIDAE

ERPOBDELLA PUNCTATA
ARTHROPODA
CRUSTACEA

ISOPODA (SOW BUGS)
ASELLIDAE

ASELLUS SP.
ASELLUS COMMUNIS
AMPHIPODA (SCUDS)
HYALELLIDAE
HYALELLA SP.
HYALELLA AZTECA
GAMMARIDAE

GAMMARUS SP.
GAMMARUS FASCIATUS
ARACHNIDA

ACARI HYDRACARINA (WATER MITES)

4. ON
4. ON

4. ON

5
2

2
4
4
5
3
4

OB
5N

5N
OB
OB
OB
ON
ON

4. OB

5. OB
5. OB
5. OB
5. OB
5. OB
5. ON
5. ON
5. ON

4. ON

4. ON
4. ON

4. ON

3. ON

2.5N
1.0N

15

TAXON NAME

INSECTA

EPHEMEROPTERA (MAYFLIES)
SIPHLONURIDAE
AMELETUS SP.
SIPHLONURUS SP.
BAETIDAE

BAETIS
BAETIS
BAETIS
BAETIS
BAETIS
BAETIS
BAETIS
BAETIS
BAETIS

SP.
SP.
SP.
SP.

1 (2 CAUDAL FILAMENTS)

2 (SHORT TERMINAL FILAMENT)

3 (3 CAULDAL FILAMENTS)
FRONDALIS

INTERCALARIS
LEVITANS

CAROLINA

PHOEBUS
VAGANS

CALLI BAETIS SP.

CENTROPTILUM SP.

CLOEON SP.

PSEUDOCLOEON SP.

PSEUDOCLOEON NR.
OLIGONEURIDAE

ISONYCHIA SP.
HEPTAGENIIDAE

EPEORUS (IRON) SP.

HEPTAGENIA SP.

HEPTAGENIA HEBE/APHRODITE

HEPTAGENIA MACULIPENNIS

HEPTAGENIA PULLA

RHITHROGENA SP.

STENONEMA SP.

STENONEMA

STENONEMA

STENONEMA
• STENONEMA

STENACRON

STENACRON

STENACRON
EPHEMERELLIDAE

ATTENELLA SP.

ATTENELLA ATTENUATA

DANNELLA SP.

DANNELLA SIMPLEX

DRUNELLA SP.

EPHEMERELLA SP.

EPHEMERELLA DOROTHEA

EPHEMERELLA INVARIA

EPHEMERELLA NEEDHAMI

EURYLOPHELLA SP.

SERRATELLA DEFICIENS
TRICORYTHIDAE

TRICORYTHODES SP.
CAENIDAE

BRACHYCERCUS SP.

MEDIOPUNCTATUM

MODESTUM

PUDICUM

PULCHELLUM

SP.

INTERPUNCTATUM S.S.

INTERPUNCTATUM FRONTALE

TOLERANCE
VALUE

2.5N

0
2
2
2
2
2
2
2
3
2
2

OH
OH
5N
5N
5N
5N
5N
OH
OH
5N
5N

1.0H
3. OH
1.0H
2. OH
2. ON
1.0H

2. OH

0.0 J

1.0H
2.0 J
0.0H
0.0H
2. ON

2. OH
0.0H
0.5N
1.0H

3. OH
3. ON

1.0H
1.0H
1.0H
0.0H

1
0
1
1
2

OH
OH
OH
OH
ON

1.0H
2. OH
2. OH

16

AQUATIC MACROINVERTEBRATE RAPID BIOASSESSMENT

TAXON NAME

CAENIS SP.
LEPTOPHLEBIIDAE

CHLOROTERPES SP.

HABROPHLEBIODES SP.

HABROPHLEBIODES AMERICANA

LEPTOPHLEBIA SP.

PARALEPTOPHLEBIA SP.
POTAMANTHIDAE

POTAMANTHUS SP.
EPHEMERIDAE

EPHEMERA SP.

EPHEMERA SIMULANS

EPHEMERA VARIA

HEXAGENIA SP.

HEXAGENIA LIMBATA
ODONATA (DRAGONFLIES AND DAMSELFLIES)
ANISOPTERA (DRAGONFLIES)
CORDULEGASTRIDAE

CORDULEGASTER SP.

CORDULEGASTER^ MACULATA
CORDULIIDAE
GOMPHIDAE

DROMOGOMPHUS SP.

GOMPHUS SP.

OPHIOGOMPHUS SP.
AESCHNIDAE

AESCHNA SP.

BASIAESCHNA SP.

BAS I AESCHNA JANATA

BOYERIA SP.

BOYERIA VINOSA
MACROMIIDAE

MACROMIA SP.
LIBELLULIDAE

PERITHEMIS SP.

PLATHEMIS SP.

PLATHEMIS LYDIA
ZYGOPTERA (DAMSELFLIES)
CALOPTERYGIDAE

AGRION SP.

CALOPTERYX SP.

CALOPTERYX MACULATUM
LESTIDAE

LESTES SP.

LESTES RECTANGULARIS

LESTES VIGILAX
COENAGRIONIDAE

ARGIA SP.

ARGIA MOESTA

ENALLAGMA SP.

ENALLAGMA CIVILE

ENALLAGMA EXSULANS

ENALLAGMA GEMINATUM

TOLERANCE
VALUE

3. OH

2

1
2

2

1

OJ
OJ
OH
OH
OH

2. OH

1.0H
1.0 J
3. OH
3.0 J

1.0H

3.0 J

2. OH
1.0H

3. OH
2. OH
1.0H
2.0 J
2.5N
2.5N

2. OH

2. OH

3. OH
3. OH
3. OH

2.5N

2. OH

3. OH
3. OH
3. OH
3. OH

17

TOLERANCE

TAXON NAME VALUE

ENALLAGMA HAGENI-EBRIUM GROUP 3 . OH

ENALLAGMA SIGNATUM 3 . OH
ISCHNURA SP.

ISCHNURA VERTICALIS 4 . OH
PLECOPTERA (STONEFLIES)

PTERONARCIDAE

PTERONARCYS SP. 1 . OH

NEMOURIDAE
NEMOURA SP.

NEMOURA VENOSA 0 . OH

LEUCTRIDAE

LEUCTRA SP. 0.0H

PERLIDAE

ACRONEURIA SP. 0 . OH

ACRONEURIA ABNORMIS 0 . OH

ACRONEURIA NR. CAROLINENSIS 0 . OH

ACRONEURIA GEORGIANA 0.0H

ACRONEURIA LYCORIAS 0.0H

NEOPERLA SP. 2 . 0 J

NEOPERLA CLYMENE 1.0H

PARAGNETINA SP. 1 . ON
PERLESTA SP.

PERLESTA PLACIDA 2 . OH
PHASGANOPHORA SP.

PHASGANOPHORA CAPITATA 0.0H
HEMIPTERA (TRUE BUGS)

CORIXIDAE (WATER BOATMEN) 4 . ON
MEGALOPTERA (DOBSONFLIES & ALDERFLIES)

SIALIDAE (ALDERFLIES)

SIALIS SP. 2. OH

CORYDALIDAE (DOBSONFLIES) 2. OH

CHAULOIDES SP. 2 . OH
CORYDALUS SP.

CORYDALUS CORNUTUS 2 . OH

NIGRONIA SP. 1.0N
NEUROPTERA

SISYRIDAE (SPONGILLA FLIES)

SISYRA SP. 1.0N

TRICHOPTERA (CADDISFLIES) 2 . 5N

PHI LOPOTAMI DAE 1 . 5N

CHIMARRA SP. 1 . 5N

CHIMARRA ATERRIMA 2 . OH

CHIMARRA OBSCURA 2 . OH

PSYCHOMYIIDAE

PSYCHOMYIA SP. 2 . ON

POLYCENTROPIDAE 2 . 5N
CERNOTINA SP.

NEURECLIPSIS SP. 4 . OH

NYCTIOPHYLAX SP. 1 . OH

PHYLOCENTROPUS SP. 2 . OH

PHYLOCENTROPUS PLACIDUS 1.0H

HYDROPS YCHI DAE 2 . 5N

CHEUMATOPSYCHE SP. 3 . OH

18

AQUATIC MACROINVERTEBRATE RAPID BIOASSESSMENT

TOLERANCE

TAXON NAME VALUE

DIPLECTRONA SP.

DIPLECTRONA MODESTA 0 . OH

HYDROPSYCHE SP. 2 . 5N

HYDROPSYCHE BETTENI 3 . OH

HYDROPSYCHE CUANIS 3 . OH

HYDROPSYCHE SIMULANS 3 . OH

(SYMPHITOPSYCHE GROUP) 2 . 5N

HYDROPSYCHE BIFIDA 3 . OH

HYDROPSYCHE PHALERATA 1 . OH

HYDROPSYCHE RIOLA 2 . OH

HYDROPSYCHE SLOSSONAE 2 . OH

HYDROPSYCHE SPARNA 1.0H
MACROSTEMUM SP.

MACROSTEMUM ZEBRATUM 2 . OH

RHYACOPHILIDAE

RHYACOPHILA SP. 0.0H

RHYACOPHILA FUSCULA 0 . OH

GLOSSOSOMATIDAE

AGAPETUS SP. 1.0H

GLOSSOSOMA SP. 1 . OH

PROTOPTILA SP. 1.0H

HYDROPTILIDAE 3 . ON

AGRAYLEA SP. 3 . OH

HYDROPTILA SP. 3 . OH

LEUCOTRICHIA SP. 3 . OH

LEUCOTRICHIA PICTIPES 3 . OH

OCHROTRICHIA SP. 3 . OH

OXYETHIRA SP. 2 . ON

PHRYGANEIDAE

AGRYPNIA SP. 2. OH

PHRYGANEA SP. 2 . ON

PTILOSTOMIS SP. 2. ON

BRACHYCENTRIDAE

BRACHYCENTRUS SP. 0 . 5N

MICRASEMA SP. 0 . 5N

LEPI DOSTOMATI DAE

LEPIDOSTOMA SP. 1 . OH

LIMNEPHILIDAE

LIMNEPHILUS SP. 2 . OH

NEOPHYLAX SP. 2 . OH

PYCNOPSYCHE SP. 2 . OH

ODONTOCERIDAE

PSILOTRETA SP. 0 . ON

MOLANNIDAE

MOLANNA SP. 1 . OH

MOLANNA UNIOPHILA 1 . OH

HELI COPS YCHI DAE

HELICOPSYCHE SP. 2 . OH

HELICOPSYCHE BOREALIS 2 . OH

LEPTOCERIDAE

CERACLEA SP. 2 . OH

CERACLEA TARSI-PUNCTATUS 2 . OH
LEPTOCERUS SP.

19

TAXON NAME

TOLERANCE
VALUE

OECETIS
OECETIS
OECETIS
SETODES

LEPTOCERUS AMERICANUS

MYSTACIDES SP.

NECTOPSYCHE SP.

OECETIS SP.

AVARA

CINERASCENS
INCONSPICUA
SP.

TRIAENODES SP.

TRIAENODES ABA

TRIAENODES NR. MARGINATA
LEPIDOPTERA (BUTTERFLIES AND MOTHS)
PYRALIDAE

NYMPHULA SP.

PARAGYRACTIS SP.

PARAPOYNX SP.
COLEOPTERA (BEETLES)

GYRINIDAE (WHIRLIGIGS)

DINEUTUS SP. (LARVAE ONLY)

DINEUTUS CILIATUS (LARVAE ONLY)

GYRINUS SP. (LARVAE ONLY)

GYRINUS FRATERNUS (LARVAE ONLY)

GYRINUS VENTRALIS (LARVAE ONLY)
HALIPILDAE

PELTODYTES SP.
DYTISCIDAE (PREDACIOUS DIVING BEETLES)

AGABUS SP.

DERONECTES SP.
HYDROPHILIDAE (WATER SCAVENGER BEETLES)

BEROSUS SP.

PARACYMUS SP.
PSEPHENIDAE (WATER PENNIES)

ECTOPRIA SP.

ECTOPRIA NERVOSA

PSEPHENUS SP.

PSEPHENUS HERRICKI
DRYOPIDAE

HELICHUS SP.
ELMIDAE (RIFFLE BEETLES)

ANCYRONYX SP.

ANCYRONYX VARIEGATA

DUBIRAPHIA SP.

DUBIRAPHIA BIVITTATA

MACRONYCHUS SP.

MACRONYCHUS GLABRATUS

MICROCYLLOEPUS SP.

MICROCYLLOEPUS PUSILLUS

OPTIOSERVUS SP. (LARVAE ONLY)

OPTIOSERVUS FASTIDITUS (LARVAE ONLY)

OULIMNIUS SP.

OULIMNIUS LATIUSCULUS

PROMORESIA SP.

PROMORESIA TARDELLA

2
2
2
2
2
2
2
2
2
2
2

OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH

1.0H
2. OH
1.0H

2
2
2
2
2

4
4

2

2
2

2

OH
OH
OH
OH
OH

4. ON

4. ON
4. ON

ON
ON

OH
OH
ON
OH

2. OH

2. OH

2. OH

2. OH

1.0H
2. OH
2. OH
2.5N
2.5N

1.0N

20

AQUATIC MACROINVERTEBRATE RAPID BIOASSESSMENT

TAXON NAME

TOLERANCE
VALUE

STENELMIS SP. (ALL LARVAE)

STENELMIS SP. 1

STENELMIS SP. 2

STENELMIS CRENATA GR. (LARVAE ONLY)
PTILODACTYLIDAE
CURCULIONIDAE
DIPTERA (TRUE FLIES)
BLEPHARICERIDAE

BLEPHARICERA SP.

BLEPHARICERA TENUIPES
TIPULIDAE (CRANE FLIES)

ANTOCHA SP.

DICRANOTA SP.

HELIUS SP.

HEXATOMA SP.

HEXATOMA (ERIOCERA) SPINOSA

LIMONIA SP.

PEDICIA SP.

PILARIA SP.

TIPULA SP.
CULICIDAE (MOSQUITOES)

ANOPHELES SP.

CULEX SP.

CULEX PIPIENS
CHAOBORIDAE (PHANTOM MIDGES)

CHAOBORUS SP.
PSYCHODIDAE (MOTH FLIES)

PSYCHODA SP.
CERATOPOGONIDAE (BITING MIDGES)

BEZZIA SP.

CULICOIDES SP.

FORCIPOMYIA SP.

PALPOMYIA SP

PALPOMYIA/PROBEZZIA SP.

PROBEZZIA SP.
SIMULIIDAE (BLACK FLIES)

SIMULIUM SP.

SIMULIUM VENUSTUM

SIMULIUM VITTATUM
CHIRONOMIDAE (MIDGES)
TANYPODINAE

ABLABESMYIA SP.

ABLABESMYIA AURIENSIS

ABLABESMYIA JUNTA

ABLABESMYIA MALLOCHI

ABLABESMYIA PELEENSIS

ARCTOPELOPIA SP.

ARCTOPELOPIA SP. 1

ARCTOPELOPIA ALBA

CLINOTANYPUS SP.

CLINOTANYPUS SP. 3

COELOTANYPUS SP.

CONCHAPELOPIA SP.

3
2
2
3

OH
5N
5N
OH

5. ON

0.0H

2
2
3
3
3
2
2
3
2

5
3
3

4

1

3
3

3
3
4
3
2
3
3
3
3
3
3
3
3
3
3
2
3

OH
OH
OH
OH
OH
OH
OH
ON
OH

5. ON
5. ON
5. ON

4. OH

OH
ON
OH
OH
OH

OH
OH

ON
OH
OH
ON
5N
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH

21

TAXON NAME

TOLERANCE
VALUE

CONCHAPELOPIA/ARCTOPELOPIA 3 . OH

DEROTANYPUS SP. 2 . 5N

DJALMABATISTA SP. 3 . ON

GUTTIPELOPIA SP. 3 . OH

MEROPELOPIA SP. 3 . OH

NATARSIA SP. 3 . OH

NATARSIA TYPE 1 3 . OH

NATARSIA TYPE 2 3 . OH

NILOTANYPUS SP. 3 . OH

PENTANEURA SP. 2 . OH

PENTANEURA SP.l 2 . OH

PENTANEURA SP.2 2 . OH

PROCLADIUS SP. 3. OH

PSECTROTANYPUS SP. 3 . OH

RHEOPELOPIA SP. 3 . OH

TANYPUS SP. 4. OH

TELOPELOPIA SP. 3 . OH

THIENEMANNIMYIA GROUP 3 . OH

DIAMESINAE

DIAMESA SP. 2. OH

PAGASTIA SP. 2. OH

POTTHASTIA SP. 2 . OH

POTTHASTIA GAEDII GR. 2 . OH

POTTHASTIA LONGIMANUS 2 . OH

SYMPOTTHASTIA SP. 2 . OH

ORTHOCLADI INAE 3 . 5N

BRILLIA SP. 3. OH

BRILLIA FLAVIFRONS 3 . OH

BRILLIA PAR 3 . OH

BRILLIA PAR (VAR.) 3 . OH

BRILLIA SERA 3 . OH

CARDIOCLADIUS SP. 3 . OH

CARDIOCLADIUS ALBIPLUMUS 2 . OH

CARDIOCLADIUS OBSCURUS 3 . OH

CORYNONEURA SP. 2 . OH

CORYNONEURA NR. CELERIPES 2 . OH

CORYNONEURA TARIS 2 . OH

CRICOTOPUS SP. 4. OH

CRICOTOPUS/ORTHOCLADIUS SP. 4 . ON

CRICOTOPUS SP. 2 4. OH

CRICOTOPUS SP. 3 4. OH

CRICOTOPUS BICINCTUS 4 . OH

CRICOTOPUS BICINCTUS GROUP 4 . OH
CRICOTOPUS CYLINDRACEUS/FESTIVELLUS GROUP 4 . OH

CRICOTOPUS EXILUS 4 . OH

CRICOTOPUS FLAVOCINCTUS (NR. ) 4 . OH

CRICOTOPUS FUGAX 4 . OH

CRICOTOPUS INTERSECTUS GROUP 4 . OH

CRICOTOPUS JUNUS 4 . OH

CRICOTOPUS SLOSSONAE 4 . OH

CRICOTOPUS SYLVESTRIS 4 . OH

CRICOTOPUS SYLVESTRIS GROUP 4 . OH

CRICOTOPUS SYLVESTRIS/ INTERSECTUS 4 . OH

22

AQUATIC MACROINVERTEBRATE RAPID BIOASSESSMENT

TAXON NAME

TOLERANCE
VALUE

CRICOTOPUS TREMULUS

CRICOTOPUS TREMULUS GROUP

CRICOTOPUS TRIANNULATUS

CRICOTOPUS TRIFASCIA

CRICOTOPUS NR. TRIFACIATUS

CRICOTOPUS VIERRIENSIS

EUKIEFFERIELLA SP.

EUKIEFFERIELLA SP. 1/SIMILIS

EUKIEFFERIELLA SP. 2

EUKIEFFERIELLA BREHMI GROUP

EUKIEFFERIELLA BREVINERVIS

EUKIEFFERIELLA BREVICALCAR GROUP

EUKIEFFERIELLA CLARIPENNIS GROUP

EUKIEFFERIELLA CYANEA GROUP

EUKIEFFERIELLA DEVONICA GROUP

EUKIEFFERIELLA GRACEI GROUP

EUKIEFFERIELLA PSEUDOMONTANA GROUP

EUKIEFFERIELLA RECTANGULARIS GROUP

E. SIMILIS GR. (NOW CARDIOCLADIUS ALB.)

EUKIEFFERIELLA SORDENS

HETEROTRISSOCLADIUS SP.

NANOCLADIUS SP.

NANOCLADIUS NR. MINIMUS

NANOCLADIUS NR. MINIMUS

ORTHOCLADIUS SP.

ORTHOCLADIUS ANNECTANS

ORTHOCLADIUS CARLATUS

ORTHOCLADIUS OBUMBRATUS

ORTHOCLADIUS ROBACKI

ORTHOCLADIUS TYPE 3

PARACHAETOCLADIUS SP.

PARACHAETOCLADIUS SP.

PARAKIEFFERIELLA SP.

PARAMETRIOCNEMUS SP.

PSECTROCLADIUS SP.

RHEOCRICOTOPUS SP.

SYNORTHOCLADIUS SP.

THIENEMANNIELLA SP.

THIENEMANNIELLA XENA

TVETENIA SP.

TVETENIA BAVARICA GROUP

TVETENIA DISCOLORIPES GROUP
CHIRONOMINAE
CHIRONOMINI

CHIRONOMUS SP.

CHIRONOMUS DECORUS/RIPARIUS

CHIRONOMUS FUMIDUS

CHIRONOMUS REDUCTUS GR. (NR.)

CHIRONOMUS RIPARIUS

CHIRONOMUS TENTANS

CLADOPELMA SP.

CRYPTOCHIRONOMUS SP.

CRYPTOCHIRONOMUS SP. 1

4
4
4
4
4
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1

OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH

1.0H

1
3
3
3
3
3
3
1
1
2
3
2
3
2
2
2
2
2
2
3
3
5
5
5

OH
OH
OH
OH
OH
OH
OH
OB
OB
ON
OH
OH
OH
5N
OH
OH
ON
ON
ON
ON
ON
OH
OH
OH

5. OH
5. OH
5. OH
4. ON
4. OH
4. OH

23

TOLERANCE

TAXON NAME VALUE

CRYPTOCHIRONOMUS SP. 3 4 . OH

CRYPTOCHIRONOMUS SP. C 4 . OH

DICROTENDIPES SP. 4 . OH

DICROTENDIPES/GLYPTOTENDIPES 4 . OH

DICROTENDIPES MODESTUS 4 . OH

DICROTENDIPES NEOMODESTUS 4 . OH

DICROTENDIPES NERVOSUS 4 . OH

DICROTENDIPES NERVOSUS TYPE 1 4 . OH

DICROTENDIPES NERVOSUS TYPE 2 4 . OH

EINFELDIA SP. 5 . OH

ENDOCHIRONOMUS SP. 3 . OH

GLYPTOTENDIPES SP. 5 . OH

GLYPTOTENDIPES LOBIFERUS 5 . ON

HARNISCHIA SP. 4 . OH

HARNISCHIA SP. 2 4 . OH

KIEFFERULUS SP. 4 . OH

MICROTENDIPES SP. 3 . OH

MICROTENDIPES CADUCUS 3 . OH

MICROTENDIPES CAELUM 3 . OH

MICROTENDIPES PEDELLUS 3 . OH

MICROTENDIPES TARSALIS 3 . OH

PARACHIRONOMUS SP. 4 . OH

PARACHIRONOMUS ABORTIVUS 4 . OH

PARACLADOPELMA SP. 3 . OH

PARALAUTERBORNIELLA SP. 3 . OH

PARATENDIPES SP. 2 . OH

PARATENDIPES ALBIMANUS/DUPLICATUS 2 . OH

PHAENOPSECTRA SP. 4 . OH

PHAENOPSECTRA SP. 1 4 . OH

PHAENOPSECTRA PROB. DYARI 4 . OH

PHAENOPSECTRA FLAVIPES 4 . OH

PHAENOPSECTRA OBEDIENS 4 . OH

POLYPEDILUM SP. 3 . OH

POLYPEDILUM AVICEPS 3 . OH

POLYPEDILUM CONVICTUM 3 . OH

POLYPEDILUM FALLAX GROUP 3 . OH

POLYPEDILUM HALTERALE ~ 3 . OH

POLYPEDILUM ILLINOENSE 3 . OH

POLYPEDILUM OBTUSUM 3 . OH

POLYPEDILUM OPHIODES 3 . OH

POLYPEDILUM SCALAENUM 3 . OH

POLYPEDILUM SCALAENUM SP. 1 3 . OH

POLYPEDILUM SCALAENUM SP. 2 3 . OH

POLYPEDILUM SIMULANS/DIGITIFER 3 . OH

POLYPEDILUM NR. TRIGONUM 3 . OH

POLYPEDILUM TRITUM 3 . OH

PSEUDOCHIRONOMUS SP. 3 . OH

STENOCHIRONOMUS SP. 2 . OH

STICTOCHIRONOMUS SP. 3 . OH

STICTOCHIRONOMUS SP. 1 3 . OH

STICTOCHIRONOMUS DIVINCTUS 3 . OH

TRIBELOS SP. 1.0N

XENOCHIRONOMUS SP. 2 . OH

24

AQUATIC MACROINVERTEBRATE RAPID BIOASSESSMENT

TAXON NAME

TOLERANCE
VALUE

XENOCHIRONOMUS (XENOCHIRONOMUS) SP.
TANYTARSINI

CLADOTANYTARSUS SP.

CONSTEMPELLINA SP.

MICROPSECTRA SP.

PARATANYTARSUS SP.

PARATANYTARSUS BOIEMICA

PARATANYTARSUS DISSIMILIS

PARATANYTARSUS DISSIMILIS/BOIEMICA

RHEOTANYTARSUS SP.

RHEOTANYTARSUS DISTINCTISSIMUS GROUP

RHEOTANYTARSUS EXIGUUS GROUP

STEMPELLINA SP.

STEMPELLINA BAUSEI

SUBLETTE A SP.

SUBLETTEA COFFMANI

TANYTARSUS SP.

TANYTARSUS GLABRESCENS

TANYTARSUS GLABRESCENS GROUP

TANYTARSUS GUERULUS GROUP
TABANIDAE (HORSE FLIES, DEER FLIES)

CHRYSOPS SP.
ATHERICIDAE

ATHERIX SP.

ATHERIX VARIEGATA
DOLICOPODIDAE
EMPIDIDAE

CLINOCERA SP.

HEMERODROMIA SP.
EPHYDRIDAE

BRACHYDEUTRA SP.
SCATHOPHAGI DAE
MUSCIDAE

LIMNOPHORA SP.
MOLLUSCA

GASTROPODA (SNAILS)
MESOGASTROPODA
HYDROBIIDAE

AMNICOLA SP.

AMNICOLA LIMOSA
BASOMATOPHORA
LYMNAEIDAE

LYMNAEA SP.
PHYSIDAE

PHYSA SP.
PLANORBIDAE

GYRAULUS SP.

GYRAULUS PARVUS

HELISOMA SP.

HELISOMA ANCEPS

HELISOMA CAMPANULATUM

HELISOMA TRIVOLVIS
ANCYLIDAE (LIMPETS)

2
3
3
2
3
3
3
3
3
3
3
3
2
2
3
3
3
3
3
3

3
3
3
3
2
2
2

3
4
4

4
4

4
4
5

OH
ON
OH
5N
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH

3. OH

2. ON
2. OH
2. OH

OH
OH
OH
OH
ON
OH
OH

3. ON

ON
ON
ON

OB
ON

ON
ON
ON

25

TAXON NAME

FERRISSIA SP.
PELECYPODA (BIVALVES)
HETERODONTA

PISIDIIDAE (FINGERNAIL CLAMS)

TOLERANCE
VALUE

3. ON
3. ON

Tolerance Value Sources:

B =R.W. Bode, Stream Monitoring Unit, New York State Department of
Environmental Conservation, Albany, NY.

H =Hilsenhoff 1982.

J = Jones, et al. 1981.

N =R.M. Nuzzo, Technical Services Branch, Massachusetts Division of
Water Pollution Control, Westborough, MA.

26

AQUATIC MACROINVERTEBRATE RAPID BIOASSESSMENT
2.2.6 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. Jones-, J.R., B. H. Tracy, J.L. Sebaugh, D.H. Hazelwood, and M.M. Smart.
1981. Biotic Index Tested for Ability to Assess Water Quality of
Missouri Ozark Streams. Trans. Amer. Fish. Soc . 110: 627-637.

9. 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.

10. 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.

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

27

SECTION PAGE

2.3 SITE ASSESSMENT 29

2.3.1 Introduction and Purpose 29

2.3.2 Objectives 29

2.3.3 Approach 29

2.3.4 Parametric Coverage 30

2.3.5 Quantitative Data Analyses 30

2.3.6 References 32

28

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) taxonomic richness; 3) evenness; and
4) diversity (e.g., Shannon Weaver H^). 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 .

29

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.

30

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 H1 is commonly used for two
in form; and (2) it has a known variance struct
attribute, a t-test for differences in H* betwe
run. The form of the index and its variance st
Poole (1974) and are presented below.

reasons: (1) it is simple
ure. Due to the latter
en two data sets can be
ructure are taken from

H =

- £~2 " pi. In pi

S-l
"ZTT"

where

Var. H' =

/" pi. In'

i=l

pi -

2> pi. In pi

i=l

N

+ S-l

2
2N

S
Pi

N =

number of taxa

the proportion of the

total number of

individuals consisting

of the itn taxon

total number of

individuals

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

where C

S

■EZ

i=l

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

and S = as above

ni = the number of

individuals in the

• th

iLI1 species

N = 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.

31

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.

32

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

33

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.1 Phytoplankton 35

3.1.1 Definition 35

3.1.2 Objectives 35

3.1.3 Field Sampling 35

3.1.4 Laboratory Analysis 36
Sample Preservation 36
Phytoplankton Examination 37

3.1.5 Field Equipment and Supply List 42

3.1.6 Data Record Sheets 43

3.1.7 References 46

34

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 biomass 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.

35

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-term 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 (Kl) 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 M3 fixative is added to
one liter of sample and stored in the dark. [M3 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] .

36

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.

37

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.

38

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) = 100Q 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) = 1Q0Q mm3

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

39

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.,
"UIl", "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").

40

Precision Data:

PHYTOPLANKTON

1. S =

\ 4=1^1
N n-1

Where

Z(x-m)

3

4

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

Cv =

or

T 2

IL - n

m

n-1

Where Cv = Coefficient of variation

P = % standard deviation of mean =

100c

v

nF"

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.

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 +10% 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.

41

3.1.5 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories
I state vehicle, clipboard
J roof racks
[ boat trailer
[ pram, oars (and locks)
[ canoe, paddles

[ boat, motor, gas can (and line)
[ anchor, rope
life jackets, seat pads

Field Apparel

| j rain gear (jacket, pants, hat)

[ hip boots and/or chest waders
I rubber gloves

Collecting and Sampling Gear

secchi disk
[ pocket thermometer
[ photometer

tape measure
j range finder
[ plastic bucket, rope
[ plastic tubing with weight attached
[ glass and/or plastic vials
[ glass and/or plastic jars, bottles

sample preservative, fixative

Miscellaneous Items

[ USGS topographic maps

[ clipboard

field data sheets, maps

tags and labels (with elastics
or string)

[ pencils, pens

field identification manuals, keys
| dissecting kit, hand lens
[ camera, film

first-aid kit

field glasses

insect repellent
| tool kit
j cooler (s) , ice

42

PHYTOPLANKTON

3.1.6 DATA RECORD SHEETS

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MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

PHYTOPLANKTON

PHYTOPLANKTON EXAMINATION SHEET

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45

3.1.7 REFERENCES

1. Abbott, I. A. and E.Y. Dawson. 1978. How to Know the Seaweeds.
Wm. C. Brown Co. Publishers, Dubuque, Iowa. vii + 141 p.

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

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

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

5. Farlow, W.G. 1881. Marine Algae of New England and Adjacent Coast.
1969 (Reprint). Wheldan and Wesley, Ltd., New York. 210 p.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

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

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

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

46

PHYTOPLANKTON

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

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

16. 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.

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

18. Taylor, W.R. 1962. The Marine Algae of the Northeastern Coast of North
America (Second Edition). University of Michigan Press, Ann Arbor.

ix + 870 p.

19. 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.

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

21. 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.

22. 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.

23. 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.

24. 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.

47

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3 . 2 Periphyton 49

3.2.1 Definition 49

3.2.2 Objectives 49

3.2.3 Field Sampling 49

3.2.4 Laboratory Analyses 49
Log-In Procedure 49
Microscopic Analysis 49
Periphyton Examination Laboratory Equipment List 50

3 2.5 Field Equipment and Supply List 51

3.2.6 Data Record Sheets 52

3.2.7 References 55

48

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.

49

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

50

PERIPHYTON

3.2.5 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories
state vehicle, clipboard
[ roof racks
[ boat trailer
[ pram, oars (and locks)
J canoe, paddles

[ boat motor, gas can (and line)
[ anchor, rope
life jackets, seat pads

Collecting and Sampling Gear

secchi disk
[ pocket thermometer
[ photometer

tape measure
I range finder
[ plastic bucket, rope
[ glass and/or plastic vials
| glass and/or plastic jars, bottles
[ sample preservative, fixative

Field Apparel

! rain gear (jacket, pants, hat)
[ hip boots and/or chest waders
[ rubber gloves

Miscellaneous Items

[ USGS topographic maps

[ clipboard

field data sheets, maps

tags and labels (with elastics or
string)

[ pencils, pens

field identification manuals, keys
[ dissecting kit, hand lens
I camera, film
Q first-aid kit

field glasses

insect repellent

tool kit
[ cooler(s) , ice

51

3.2.6 DATA RECORD SHEETS

52

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

Identification:

Code(s)

Sample #:

Habitat

Substrate

Relative
Abundance

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.

54

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.

55

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. 1979. Mad River Press, Inc.,
Eureka, CA. 119 p.

56

AQUATIC AND WETLAND VEGETATION

SECTION

PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3. 3 Aquatic and Wetland Vegetation

3.3.1 Definition

3.3.2 Objectives

3.3.3 Field Sampling

3.3.4 Laboratory Analyses

3.3.5 Field Equipment and Supply List

3.3.6 Data Record Sheets

3.3.7 References

58
58
58
58
58
59
60
69

57

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.

58

3.3.5 FIELD EQUIPMENT AND SUPPLY LIST

AQUATIC AND WETLAND VEGETATION

Vehicles, Boats and Accessories
j state vehicle, clipboard
J roof racks
[ boat trailer
I pram, oars (and locks)
J canoe, paddles

boat motor, gas can, (and line)
[ anchor, rope

life jackets, seat pads
Collecting and Sampling Gear

secchi disk
J pocket thermometer
[ photometer

tape measure
j range finder
j plastic bucket, rope
[ glass and/or plastic vials
j glass and/or plastic jars, bottles
( plastic bags (and ties)
j sample preservative, fixative
I rake

[ grappling hook, rope
j Ekman, Ponar dredges
[ white enamel trays
j trowel
[ plant press and vasculi

Field Apparel

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

Miscellaneous Items

[ USGS topographic maps

[ clipboard

field data sheets, maps

tags and labels (with elastics or
string)

[ pencils, pens

field identification manuals, keys
I dissecting kit, hand lens

first-aid kit

field glasses
j insect repellent
□ tool kit

j cooler(s) , ice

.urn

59

3.3.6 DATA RECORD SHEETS

60

AQUATIC AND WETLAND VEGETATION

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MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

AQUATIC MACROPHYTE CODE

Macroscopic algae (mats, clumps, etc.)
Bryozoan

Chara sp. "Muskgrass"
Nitella sp. "Stonewort"

Moss

Riccia fluitans "Slender Riccia"

Ricciocarpus natans "Purple-fringed Riccia'

CI
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Marsilea quadrifol ia "Pepperwort"
Azolla caroliniana "Water-velvet"
Salvinia rotundifolia "Floating Moss"
Other aquatic ferns

Isoetes sp. "Quillwort"

I. Tuckermani "Quillwort"

I

II

Typha latifolia "Common Cattail"

Typha angustifolia "Narrow-leaved Cattail"

T
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Sparganium sp. "Bur Reed"

S_. f luctuans "Water Bur Reed"
S_. eurycarpum "Giant Bur Reed"
S. americanum "Bur Reed"

S

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S2
S3

Potamogeton sp. "Pondweed"

P_. amplifolias "Largeleaf Pondweed"

P_. crispus "Curlyleaf Pondweed"

P. Richardsonii "Richardson Pondweed"

P

PI
P2
P3

63

P_. Robbinsii "Flatleaf Pondweed" P4

P_. epihydrus "Ribbonleaf Pondweed" P5

_P. sp. "Thin-leaved Pondweed" P6

P_. gramineus "Grassleaf Pondweed" P7

P_. natans "Floatingleaf Pondweed" P8

P_. Vaseyi "Vasey's Pondweed" P9

P_. capil laceus "Pondweed" P10

P_. fol iosus "Leafy Pondweed" Pll

F\ tenuifolius "Pondweed" P12
P_. perfol iatus "Redhead Grass" or "Thorowort Pondweed" P13

P_. pusi 1 1 us "Slender Pondweed" or "Baby Pondweed" P14

P_. Spirillus "Snail seed Pondweed" P15
P_. pectinatus "Sago Pondweed" or "Fennel leaf Pondweed" P16

P.. illinoensis "Illinois Pondweed" P17

P. pulcher "Heartleaf Pondweed" P18

P_. bicupulatus "Snail seed Pondweed" P19

P_. zosteriformis "Flatstem Pondweed" P20

P. nodosus P21

Najas sp. "Bushy Pondweed" or "Naiad" J

Ruppia maritima "Widgeon Grass" Jl

Najas Flexilis "Slender Naiad" J2

Alisma sp. "Water-Plantain" Al

Echinodorus sp. "Burhead" A2

Sagittaria sp. "Arrowhead" or "Duck Potato" A3

Sagittaria sp. (submerged form only) A4

S_. latifol ia "Common Arrowhead" A5

S. rigida "Stiff Arrowhead" A6

S. teres "Dwarf Wapato" A7

S^. graminea "Grassy Arrowhead" A8

Vallisneria americana "Wild Celery" or "Tape Grass" Hi

Elodea sp. "Waterweed" H2

E. Nuttallii "Waterweed" H3

E_. canadensis "Canadian Waterweed" H4

Gramineae (Grass Family) • G

64

AQUATIC AND WETLAND VEGETATION

Cyperus sp. "Flat Sedge' Yl

Dul ichium arundinaceum "Three-way Sedge" Y2

Fimbristylis sp. "Fimbn'styl is" Y3

Rynchospora sp. "Beak Rush" Y4

Cladium sp. "Twig Rush" or "Sawgrass" Y5

Carex sp. X

Scirpus sp. "Bulrush" B

S. validus "Softstem Bulrush" Bl

£. cyperinus "Wool grass Bulrush" B2

_S. americanus "American Bulrush" B3

S. atrovirens "Dark-green Bulrush" B4

Eleocharis sp. "Spike Rush" E

E. acicularis "Needle Spike Rush" El

E. Small ii "Spike Rush" E2

E_. palustris "Common Spike Rush" E3

Peltandra virginica "Arrow Arum" al

Call a palustris "Water Arum" a2

Orontium aquaticum "Golden Club" a3

Acorus Calamus "Sweet Flag" a4

Spirodela polyrhiza "Big Duckweed" Ll

Wolffia sp. "Watermeal" L2

Wolffiella floridana "Florida Wolff iella" L3

Lemna sp. "Duckweed" L4

L_. minor "Common Duckweed" L5

l_. trisulca "Star Duckweed" L6

Xyris sp. "Yellow-eyed Grass" e

Eriocaulon sp. "Pipewort" el

E. septangulare "Pipewort" e2

Heteranthera dubia "Mud Plantain" Wl

Pontederia cordata "Pickerelweed" W2

P. cordata forma taenia "Pickerelweed" W3

65

Iris sp. "Iris" jl

Juncus sp. "Rush" j2

Soururus cernuus "Lizard's Tail" j3

Rumex sp. "Dock" Ql

Polygonum sp. "Smartweed" Q2

Sal ix sp. "Willow" bl

Myrica Gale "Sweet Gale" b2

Alnus sp. "Alder" b3

Nyssa sp. "Sour Gum" or "Tupelo" b4

Cornus sp. "Dogwood" b5

Chamaedaphne calyculata "Leatherleaf ' b6

Fraxinus sp. "Ash" b7

Cephalanthus occidental is "Buttonbush" b8

Ilex verticillata "Virginia Winterberry" or "Black Alder" b9

Clethra alnifol ia "Sweet Pepperbush" blO

Ceratophyllum demersum "Coontail" K

Nymphaea sp. "Water Lily" Nl

j^. odor at a "Fragrant Water Lily" N2

N. tuberosa "White Water Lily" N3

Nuphar sp. "Yellow Water Lily", "Cow Lily", or "Spatterdock" N5

H_. variegatum "Painted Cow Lily" N6

Brasenia Schreberi "Water Shield" nl

Nelumba lutea "American Lotus" n2

Cabomba carol iniana "Fanwort" n3

Caltha palustris "Marsh Marigold" Rl

Myosurus minimus "Mousetail" R2

Ranunculus sp. "Buttercup" or "Crowfoot" R3

Subularia aquatica "Awlwort" Ml

Neobeckia aquatica "Lake Cress" M2

Cardamine sp. "Bitter Cress" M3

Rorippa sp. "Water Cress" M4

66

AQUATIC AND WETLAND VEGETATION

Podostenum sp. "River Weed"

Cal 1 itriche sp. "Water Starwort" kl

Elatine sp. "Waterwort" k2

Viola sp. "Violet" k3

Hypericum sp. "St. John's-wort" • — k4

H. boreale f. call itrichoides "St. John's-wort" k5

Decodon vertici 1 latus "Swamp Loosestrife" VI

Trapa natans "Water Chestnut" V2

Ludwigia sp. "False Loosestrife" V3

Lythrum Sal icaria "Spiked Loosestrife" V4

Rhexia virginica "Virginia Meadow-beauty" V5

Hippuris vulgaris "Mare's-tail " hi

Prosperinaca sp. "Mermaid Weed" h2

Myriophyl lum sp. "Water Milfoil" h3

M. heterophyllum "Broad! eaf Water Milfoil" h4

M. humile "Water Milfoil" h5

M. tenellum "Leafless Milfoil" h6

Si urn suave "Water Parsnip" fl

Hydrocotyle sp. "Water Pennywort" f2

Cicuta sp. "Water Hemlock" f3

Hottonia inflata "Featherfoi 1 " ml

Samolus sp. "Water Pimpernel" m2

Lysimachia sp. "Loosestrife" m3

L_. cil iata "Loosestrife" m4

Nymphoides cordatum "Floating Heart" gl

Asclepias sp. "Milkweed" g2

Myosotis sp. "Forget-me-not" g3

Stachys sp. "Hedge Nettle" tl

Scutellaria sp. "Skullcap" t2

Physostegia sp. "False Dragonhead" t3

Lycopus sp. "Water Horehound" t4

67

Mentha sp. "Mint" t5

Solanum Dulcamara "Nightshade" t6

Utricularia sp. "Bladderwort" U

U_. vulgaris "Common Bladderwort" Ul

U. purpurea "Purple Bladderwort" *■" U2

U. inflata "Floating Bladderwort" U3

U. intermedia "Flat-leaved Bladderwort" U4

Bacopa sp. "Water Hyssop" Fl

Limosella sp. "Mudwort" F2

Veronica sp. "Speedwell" F3

Chelone sp. "Turtlehead" F4

Mimulus sp. "Monkey Flower" F5

Lindernia sp. "False Pimpernel" F6

Gratiola sp. "Hedge Hyssop" F7

G. virginiana "Hedge Hyssop" F8

Lobelia sp. 0

L_. cardinalis "Cardinal Flower" 01

L. Dortmanna "Water Lobelia" 02

Megalodonta Becki i "Water Marigold" Zl

Eupatorium sp. "Joe-pye Weed" Z2

Bidens sp. "Bur Marigold", "Beggar-ticks", or "Pitchforks" Z3

Helenium sp. "Sneezeweed" Z4

Sol idago sp. "Goldenrod" Z5

Aster sp. "Aster" Z6

Coreopis rosea "Pink Tickseed" Z7

Equisetum sp. "Horsetail" i

E_. fluviatile "Swamp or Water Horsetail" il

Drosera rotundifol ia "Roundleaf Sundew" D

Vaccinium sp. "Cranberry" d

Phragmites sp. "Reed Grass" q

68

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.

69

12. Fair-brothers, 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.E. Crow. 1982. Aquatic Vascular Plants of New
England: Part 5. Araceae, Lemnaceae, 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.

70

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 Keys. Studies in Botany Number 11.
Oregon State College, Corvallis. vi + 184 p.

31. Tiner, R.W. , Jr. 1988. Field Guide to Nontidal Wetland Identification,
Maryland Department of Natural Resources, Annapolis, Maryland and U.S.
Fish and Wildlife Service, Newton Corner, Massachusetts. xvi + 283 p.
+ plates.

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

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

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

35. 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.

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

37. 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.

71

SECTION PAGE
3.0 BIOLOGICAL FIELD AND LABORATORY METHODS
3.4 Aquatic Macroinvertebrates

3.4.1 Definition 73

3.4.2 Objectives 73

3.4.3 Field Sampling 73
Qualitative 73
Rapid Assessment 73
Quantitative 74

3.4.4 Laboratory Analysis 74

3.4.5 Instructions for Mounting Midge Larvae and Pupae 76

3.4.6 Instructions for Labeling Specimens 78

3.4.7 Field Equipment and Supply List 79

3.4.8 Data Record Sheets 80

3.4.9 References 84

72

AQUATIC MACROINVERTEBRATES
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 Macroinvertebrate Rapid Bioassessment (MRB),
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

73

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 MRB 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 without
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

74

AQUATIC MACRO INVERTEBRATES

the dish is moved to 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. Extracted specimens are stored
in 70% 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. Each vial of specimens and each slide of mounted
material must be labeled as described under "Instructions For Labeling
Specimens ."

75

3.4.5 INSTRUCTIONS FOR MOUNTING MIDGE LARVAE AND PUPAE

INSTRUCTIONS FOR MOUNTING MIDGE LARVAE

1) Place larvae in a watch glass along with a sufficient volume of 70% alcohol
to keep them covered;

2) Under low power on a dissecting scope, group the larvae according to size
and general appearance. This usually results in midges of the same tribe
or subfamily being mounted together, which in turn will aid in working
specimens through the keys more quickly;

3) Place a label on one end of a 25 x 75 mm glass microscope slide. With the
label to the right, place three similar-sized larvae together on the slide,
all with their heads pointed down;

4) Add enough CMC-10 to surround the specimens. Spread out the mounting medium
so it doesn't sit in a "bubble" around the specimens;

5) Excise the head capsules, making sure that each remains associated with its
own body, and is positioned with the mouth parts (ventral side) up;

6) Place a glass cover slip (No. 1, 18 mm x 18 mm) over the specimens and,
using forceps, apply gentle pressure to force out air bubbles and flatten
the head capsules until the mouth parts spread out slightly;

7) Add additional mounting medium to the edge of the cover slip if necessary
to rid the mount of air bubbles;

8) Record any useful observations about the specimens (e.g., if ventral tubules
are present, and how many pairs) on the SLIDE INVENTORY CATALOG SHEET.
Refer to the specimens based on their position relative to the label: "A"
indicating the cover slip closest to the label and "1" indicating the
specimen closest to the label. Thus, Al is the specimen closest to the
label and B3 is the furthest (see fig. 1).

fCC

9 9

e © e

DATE
STREAM
LOCATION
SLIDE #

cover slip B cover slip A label
Figure 1. Layout of midge larvae mounted on a glass microscope slide.

9) Allow the mount to dry overnight before putting the slides into a slide box,
Also check for air bubbles creeping under the edges of the cover slips as
the medium dries. Keep the SLIDE INVENTORY CATALOG SHEET with the slide
box so that the notes on the specimens are handy while keying out the
organisms, and for recording the identifications.

76

AQUATIC MACROINVERTEBRATES

INSTRUCTIONS FOR MOUNTING MIDGE PUPAE

1) Place midge pupae in watch glass along with 70% ethanol and examine
under a dissecting microscope;

2) Record notes of markings, the form of the thoracic horn (if present),
and the form of the case (if any) around the pupa (Rheotanytarsus spp.
larvae and pupae can be recognized quickly from the case);

3) If the larval skin and head capsule are not still attached to the pupal
body the pupa should be cleared before mounting (if larval skin, with
larval head intact, is present, skip to next step). Clear the pupa by
"cracking" the dorsal thoracic suture with a fine insect pin (0 or 00)
and immersing the specimen in 10% KOH for two hours (if there is more
than one pupal form present, clear each in a separate vial). After the
two hours rinse the pupa in distilled water and place in 70% ethanol;

4) With the labeled end of a 25 x 75 mm slide to the right, place a pupa on
the slide with ventral side down, with its head pointing toward the
bottom edge of the slide. Cover specimen with CMC-10 mounting medium.
Using an insect pin, carefully detach the head from the body, and
position the head ventral side up. If the larval head capsule is
included, mount it next to the pupa with the ventral side up. Spread
the wing sheaths out to the sides of the thorax. Drop cover slip into
place and gently force out air bubbles, but avoid "squashing" the pupa
anymore than absolutely necessary. Mount only one individual per cover
slip, but two cover slips per slide (see fig. 2);

sjb

d8/

DATE
STREAM
LOCATION
SLIDE #

cover slip B cover slip A

label

Figure 2. Layout of midge pupae mounted on a glass microscope slide

5) Apply additional mounting medium to the edges of the cover slips if
required to remove air bubbles. After 24 hours of drying examine the
mount for air under the cover slips caused by shrinkage of the mounting
medium, add mounting medium as required.

6) Be sure the record notes about specimen directly onto the SLIDE
INVENTORY CATALOG SHEET.

77

3.4.6 INSTRUCTIONS FOR LABELING SPECIMENS

1) All vials and slides containing macroinvertebrate specimens from TSB
biomonitoring surveys will be labeled immediately;

2) Each label will contain the following minimum information:

a) biomonitoring tracking number

b) station code

c) date of collection

d) name of stream organism was collected from

e) location (town, state, nearest road);

3) Additionally, each vial label will list the name of the organisms con-
tained inside the vial, whereas the labels placed on slides will show
the number of the slot it occupies in the slide storage box;

4) Labels for use in vials will be cut to an appropriate size from unlined
index cards. These will be placed inside the vial, and therefore must
be written out with pencil or black India ink. As a convenience for
locating and reading information, all the information listed in 2 above
will be shown on one side of the label, and the left edge of the label
will be placed in the vial first. The sequence for filling in
information on the vial label is shown in the example in figure 3;

5) Stick-on labels will be used for microscope slides. Information will be
written on the label such tht it appears right side up when the slide is
in place on the stge of the microscope with the label to the right-as
shown in the example in figure 4.

Survey code,
stream no-t*ie

87-5- /V)!/*? 17" June /?87

Otte r K i v/e r ; fit 2fl} GurJner, MA

in vi*L

Figure 3. Example of specimen label to be placed inside a vial.

.survey coA* i- si-i'***

87-5"- AU H

Otter River
!We 2.0X

I

■stnea.m nocme

• s£*<ie cecorA &-

Figure 4. Example of label to be placed on microscope slide mount

78

AQUATIC MACROINVERTEBRATES

3.4.7 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories
[ state vehicle, clipboard
[ roof racks
[ boat trailer
[ pram, oars (and locks)
[ canoe, paddles
I I boat motor, gas can (and line)
[ anchor, rope
life jackets, seat pads

Collecting and Sampling Gear
j_ [ pocket thermometer

! tape measure

[ range finder
I I Ekman, Peterson, Ponar dredges

[ Surber samplers

[ Hess sampler

{ metal holding tub

J white enamel trays
I | sieves (of various sizes)
I I plastic bucket, rope
I I glass and/or plastic vials

I I glass and/or plastic jars, bottles

I I ethanol, formalin

I | killing jar, killing agent

I I aerial net, D-frame net

Field Apparel

I I rain gear (jacket, pants, hat)

I I bip boots and/or chest waders
[ rubber gloves

Miscellaneous Items

I I USGS topographic maps

[ clipboard

I I field data sheets, maps

I I tags and labels (with elastics or
string)

I [ pencils, pens

I I field identification manuals, keys

I I dissecting kit, hand lens
I | camera, film

I | first-aid kit

I I field glasses

I | insect repellent

□ tool kit

[j cooler(s), ice

79

3.4.8 DATA RECORD SHEETS

80

AQUATIC MACROINVERTEBRATES

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81

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

AQUATIC MACROINVERTEBRATE 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

Epheraeroptera

Gastropoda
Pelecypoda

Odonata

Others

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

Total No. of Organisms
Total No. of Kinds

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

P = Pupa
A = Adult

82

AQUATIC IIACROINVERTEBRATES

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

SLIDE INVENTORY CATALOG SHEET

Page

of

SURVEY NAME:
SURVEY CODE:
SLIDE BOX OF

SLOT /STAT I ON CS TAXA

COMMENTS

SLOT/STATION CS TAXA COMMENTS

B

B

B

B

B

B

B

B

B

B

CS = cover slip

83

3.4.9 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 Hydrobiology (Freshwater Biology).
Pergamon Press, Inc., Elmsford, 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.

84

AQUATIC MACROINVERTEBRATES

8. . 1965. A Revision of the Genus Ephemerella

(Ephemeroptera: Ephemerillidae) VIII. The Subgenus Ephemerella in North
America. Misc. Publ. Entomol. Soc. Amer. 6(4): 243-282.

9. Bednarik, A.F. and W.P. McCafferty. 1979. Brosystematic Revision of the
Genus Stenonema (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 Sphaeriacean 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. Klemra. 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.

85

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. Saether, O.A. 1977. Taxonomic Studies on Chironomidae: Nanocladius ,
Pseudochironomus , and the Harnischia complex. Bulletin 196. Department
of Fisheries and the Environment, Fisheries and Marine Service, Ottawa,
viii + 143 p.

31. 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.

32. 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.

33. Smith, D.G. 1986. Keys to the Freshwater Macroinvertebrates of
Massachusetts (No. 1): Mollusca Pelecypoda (Clams, Mussels).
Massachusetts Division of Water Pollution Control, Westborough. iii +
53 p.

34. Smith, D.G. 1987. Keys to the Freshwater Macroinvertebrates of
Massachusetts (No. 2): Mollusca Mesogas tropoda (Operculate Snails).
Massachusetts Division of Water Pollution Control, Westborough. iii +
35 p.

35. Smith, D.G. 1988. Keys to the Freshwater Macroinvertebrates of
Massachusetts (No. 3): Crustacea Malacostraca (Crayfish, Isopods,
Amphipods). Massachusetts Division of Water Pollution Control,
Westborough. iii + 58 p.

36. Smith, D.G. 1989. Keys to the Freshwater Macroinvertebrates of
Massachusetts (No. 4): Benthic Colonial Phyla (Colonial Hydrozoans ,
Moss Animals). Massachusetts Division of Water Pollution Control,
Westborough. iii + 49 p.

86

AQUATIC MACROINVERTEBRATES

37. 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.

38. 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.

39. 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.

40. Stimpson, K.S., D.J. Klemm, 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.

41. 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.

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

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

44. . 1958. The Odonata of Canada and Alaska, Volume Two.

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

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

46. Wiederholm, T. (ed.). 1983. Chironomidae of the Holarctic Region. Part 1
Larvae. Supplement No. 19 Entomologica Scandinavica. Lund, Swenden.

457 p.

47. Widerholm, T. (ed.). 1986. Chironomidae of the Holarctic Region. Part 2 -
Pupae. Supplement No. 28. Entromologica Scandinavica. Lund, Sweden.
482 p.

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

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

50. 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.

87

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.5 Fish 89

3.5.1 Definition 89

3.5.2 Objectives 89

3.5.3 Field Sampling 89
Physical Measurements 89
Gill Netting 89
Electrof ishing 90
Trapping 90
Field Processing 90

3.5.4 Laboratory Analysis 90
Processing 90
Aging 91

3.5.5 Data Management 91
Reporting of Results 91
Computer Files 91

3.5.6 Field Equipment and Supply List 92

3.5.7 Data Record Sheets and Freshwater and Anadromous Fishes 93
Coding List

3.5.8 References 100

88

FISH

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 complimentary 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.

89

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. Gill nets are checked every two hours to minimize the
number of unwanted fish collected. When an adequate sample size is not
obtained during a typical one day set, occasionally large meshed gill
nets are reset and left overnight.

Electrof ishing

Electrof ishing is a sampling technique in which an electric current,
either alternating (a.c.) or direct (d.c), is generated into the water
to temporarily stun fish for subsequent capture. To meet sampling
needs, two types of electrof ishing are employed depending on the site
specific situation. In areas with an adequate boat access and water
deep enough for outboard motor use, an electroshock boat utilizing a gas
operated generator is used. In smaller lotic situations with a bottom
substrate and depth suitable for wading, a battery operated backpack
electrof ishing unit is applied. Using either method only those fish
appropriate to the sampling scheme are netted and retained until an ade-
quate sample size is obtained.

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.

Field Processing

Fish are sampled using any combination of the previously mentioned tech-
niques. Sampling is continued until sampling goals are met or until
time becomes a constraining factor. All fish are kept intact and fresh
in a cooler of ice and transported back to the TSB lab for further pro-
cessing and preparation.

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. 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

90

FISH

of the fish. The fillet is then placed, skin down, on the glass
filleting surface. The knife is used to separate the flesh from the
skin. Skin is discarded except when preparing trout (Salmonidae ) . Skin
is left intact on trout because it is believed to be the most common
preparation method used by fishermen. One fillet, depending on the
study, is either wrapped 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 , percent
lipids, and organic scan 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 filleting surface
and knife are rinsed thoroughly after each fish is filleted. Processed
fish are kept frozen until they are transported to the analytical
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+, 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 DBase3+ 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.

91

3.5.6 FIELD EQUIPMENT AND SUPPLY LIST

Vehicles, Boats and Accessories
[ state vehicle, clipboard
[ electrof ishing boat
[ boat motor, gas can (and line)
I generator and gas can
[ generator tote barge
[ anchor, rope

life jackets, seat pads

fire extinguisher
[ boat lights

Collecting and Sampling Gear
[ backpack electrof ishing gear
[ pocket thermometer

tape measure
[ range finder
[ plastic bucket, rope
[ plastic bags (and ties)
[ glass and/or plastic vials
[ glass and/or plastic jars, bottles
[ formalin
[ dip-nets
[ gill nets

fish measuring board
[ pan balance

Field Apparel

[ rain gear (jacket, pants, hat)
[ hip boots and/or chest waders
I rubber gloves

Miscellaneous Items

I USGS topographic maps

[ clipboard

j field data sheets, maps

length-weight, length-frequency forms

tags and labels (with elastics or
string)

[ pencils, pens

field identification manuals, keys
[ dissecting kit, hand lens
[ aluminum foil and plastic wrap
[ camera, film

first-aid kit

field glasses
[ insect repellent
[ tool kit
[ cooler(s), ice
| paper towels
[ flashlights
[ ear protectors

92

FISH

3.5.7 DATA RECORD SHEETS AND FRESHWATER AND ANADROMOUS FISHES

CODING LIST

93

MASSACHUSETTS DIVISION OF WATER POLLUTION CONTROL
TECHNICAL SERVICES BRANCH

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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.

100

CAGED MINNOW TOXICITY ASSESSMENTS

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.6 Caged Minnow Toxicity Assessments 102

3.6.1 Definition 102

3.6.2 Objectives 1°2

3.6.3 Field Sampling 102
Selecting and Setting Test Stations 102
Minnow Cage Construction 103
Study Methods 103

3.6.4 Field Equipment and Supply List 107

3.6.5 Data Record Sheet 109

3.6.6 References 111

101

3.6 CAGED MINNOW TOXICITY ASSESSMENTS

3.6.1 DEFINITION; The acute impacts of both point and non-point source
discharges to lotic environments can be evaluated with instream toxicity
studies using caged test organisms. Upstream or control stations are
necessary for comparing background levels of toxicity against levels
detected at test stations. Where applicable, laboratory toxicity testing
of the discharge is also recommended for the overall toxicity assessment.
Although limited by length of exposure, for the purpose of this Standard
Operating Procedure, caged minnow toxicity assessments are conducted with
less than 14-day-old caged fathead minnows (Pimephales pjromelas) placed
instream for a 24-hour period. A minimum of two cages/station with each
cage containing 10 minnows is required. The number of surviving minnows
at the end of the 24-hour study is recorded for each cage. The mean
percent mortality at each station is considered the measure of instream
acute toxicity.

3.6.2 OBJECTIVES

1. To characterize the magnitude and extent of acute effects of point
or non-point source discharges on fathead minnows held in the receiving
stream ( s) ;

2. to provide ambient monitoring data for the 305(b) and
304(1) reports; and

3. to provide a screening tool for assessing the water quality of the
Commonwealth .

3.6.3 FIELD SAMPLING

Selecting and Setting Test Stations

Instream test station locations are selected with regard to the magnitude
and nature of the discharge as well as results of a mixing zone
determination via an effluent dye study. In some situations, pertinent
chemical sampling (e.g., amperometric titrations for Total Residual
Chlorine [TRC] in /^g/1 downstream of a chlorinated discharge) may also
be necessary for the station location process. Finally, physical
characteristics such as wadable segments and velocity of the receiving
stream will also dictate actual station locations.

After test stations are selected, distance from the discharge to each
station is measured by either tape or rangefinder. Streamside trails
may be cut where necessary to ease access to each station if a rigorous
sampling schedule is planned.

Instream control and test stations are set with a pair of steel

102

CAGED MINNOW TOXICITY ASSESSMENTS

reinforcing rods pounded into the stream bottom. The rods are aligned
parallel with the stream flow approximately 0.5m apart and serve as
anchors for the minnow cages. If necessary, cinder blocks can be stacked
instream just below the instream test station and a plank placed from the
stream edge onto the blocks. Water quality monitoring can then be
conducted at each instream test station from the plank without disturbing
the sediments. A dipper may be used to obtain water samples from the
stream bank.

Minnow Cage Construction

Minnow cages consist of a composite of two containers; one nested inside
of the other (see Figure 1). The inner container holds the minnows and
is constructed with 5cm diameter acrylic plastic tube (plexiglass) with
0.5mm mesh Nytex™ screen attached to each end following the design of
O'Brien and Kettle (1981). The outer container is used to reduce the
water velocity flowing through the inner cage, thus protecting minnows
from mechanical stress. Bottle caps may also be used to further reduce
stream velocity. Outer containers are constructed from perforated, 1-
liter seltzer water or soda bottles with removable bottoms. The inner
cage is placed inside the outer container, after which the bottom of the
latter is attached and held in place with rubber bands and paper clips.

Study Methods

The instream waste concentration of the effluent or non-point source
discharge should be determined as part of the instream toxicity
assessment. Flow measurements should be taken above and below the
discharge as described in the Basins Program Standard Operating
Procedures (Technical Services Branch, 1989). Alternatively, instream
chloride data can be used to calculate the instream waste concentration
of the discharge (Szal et al., in preparation).

Time of travel to each instream test station is determined by dosing
the effluent or a specific stream reach with rhodamine dye and recording
the time at which the visible leading edge of the dye reaches each test
station. This information can be used to determine site specific
sampling times as well as persistence of certain wastewater constituents.

It is convenient to transport the minnows from the laboratory to the
test site in a plastic bag placed in a styrofoam cooler (Jones, 1985).
Minnows must be acclimated to ±1°C of the stream temperature as measured
with a precalibrated hand-held thermometer. According to EPA methods,
aquatic organisms should not be subjected to more than a 3°C shift in
temperature over a 12-hour period (Peltier, 1978). This may be
accomplished by bathing the minnow container in a cooler containing
stream water until temperature equilibration is realized without
exceeding the above-mentioned change in temperature. The temperature-
acclimated minnows should then be randomly transferred into the inner
test cages whicb have been set into a porcelain pan filled with
approximately one inch of stream water. Minnows can either be scooped
up into a small cup or caught with a wide-bore disposable pipet and
distributed two or three at a time to each inner cage until all cages
contain 10 minnows. Sluggish or damaged minnows should be replaced to

103

Figure 1. Minnow Cage Construction.

Inner and outer sections of the minnow cages used in the field
experiments are depicted. The outer section, which is used to slow the
velocity of water passing over the inner cage, is made from an 1-liter,
plastic soft drink bottle. Inner cages with minnows are placed inside
the top section of the outer cage; the latter is pushed into the bottom
section and held in place with elastic bands and paper clips.

Inner cages are constructed from acrylic plastic tube (plexiglass)
and Nytex^ screen. The two halves of the inner cage are held
together by elastic bands fitted over small plexiglass blocks which are
glued to each half of the inner cage. A plexiglass sleeve is placed
around the joint of the two halves to prohibit minnows from escaping
should one of the elastics fail.

Outer Cage

bottom

top

7

elastic

elastic

6.3 cm.

Inner
Cage

0-5 mm.
mesh

I ^ 4 cm. >|

From Szal et al. , in preparation

104

CAGED MINNOW TOXICITY ASSESSMENTS

insure quality control. Minnows should be counted twice before the top
half of the inner cage is attached with rubber bands and the sleeve is
positioned correctly (see Figure 1).

The porcelain pan is then submerged into a cooler containing stream
water to fill the inner minnow cages with water. Air bubbles trapped
against the Nytex™ screen are gently removed by tapping. Each inner
cage is placed into an outer container which is subsequently secured
with rubber bands and paper clips under water. Care must be taken to
keep the minnow cages submersed at all times to prevent the minnows from
becoming impinged upon the mesh screens.

Starting at the upstream or control station and successively working
downstream, the field crews deploy a minimum of two minnow cages at each
test station. The cages are wired between the rebar at a preselected
distance below the stream surface (e.g., six inches). Time and date of
minnow deployment is recorded on the Minnow Recording Sheets (see page
110) for each station. Several measurements are also conducted at the
initiation of the toxicity test.

Temperature is taken with a precalibrated hand-held thermometer, while
pH is measured electrometrically. A minimum of three velocity
measurements taken with a current meter placed adjacent to the minnow
cages are also recorded. Site specific sampling (e.g., TRC titrations,
water quality sampling) may also be conducted at this time. Dissolved
oxygen (DO) samples are also taken at each station, fixed, and titrated
back at the laboratory according to a modification of the Azide
Modification of the Winkler Method (Rand, 1975) as described in the
Basins Program Standard Operating Procedures (Technical Services Branch,
1989). DO meters may be substituded except in the presence of
chlorinated wastewater where the membrane on the DO probe may be affected
by chlorine.

The instream toxicity assessments are terminated at the end of the 24-
hour study period. The inner minnow cage is removed under water from
the outer container. A porcelain pan is placed under the submersed
inner cage and is then removed from the stream. At this time, the
elastics, sleeve and the upper section of the inner cage are removed
and minnows are checked for survival. Minnows not responding to gentle
prodding with a pipet are considered dead. Time, date, and the number
of surviving minnows is checked and recorded on the lower half of the
Minnow Recording Sheets at each corresponding station. The same series
of measurements taken at the initiation of the toxicity study are
conducted and recorded again. Minnows should not be introduced to the
stream at the end of the toxicity assessment. The screens of the inner
cages can be cleaned with a toothbrush to remove sediment and minnow
carcasses.

The design of the water quality sampling and analysis program for each
study is determined on a site by site basis. Composite effluent samplers
may be set to collect effluent during the 24 hours of study. This
effluent sample can then be mixed, split and fixed as necessary for
selected chemical analyses and laboratory toxicity tests. In the case
of a chlorinated wastewater discharge, monitoring of TRC at instream test

105

stations may be warranted every 2 to 3 hours whereas an intermittent
industrial wastewater discharge might only require water quality
monitoring at one time during the study. Ambient toxicity monitoring
with caged fathead minnows might not require any additional water quality
samples other than temperature, pH, DO, and a measurement of stream
velocity.

106

CAGED MINNOW TOXICITY ASSESSMENTS

3.6.4 FIELD EQUIPMENT AND SUPPLY LIST
Vehicles, Boats and Accessories

I | state vehicle, clipboard, keys

I | roof racks

I | boat trailer

| I pram, oars (and locks)

| | canoe, paddles, ropes

| | boat motor, gas can (and line)

I | anchor, rope

I I life jackets, seat pads

| | brush cutter, clippers, saws

| I premixed fuel

| | work gloves

Field Apparel

I rain gear

I hip boots and/or waders

| I disposable gloves

Collecting and Sampling Gear

Caged Minnow Studies

| I pocket thermometer

I | siphon tube

I | <14 day old fathead minnows

I I tape measure

| I range finder

I I ISCO™ composite samplers

I | extension cords

I | rebar

| I sledge hammer

I | flagging

I | reflective tape

I I large bore disposable pipets

I I inner minnow cages

I I outer minnow cages

I | wire (coated)

I I elastics

I | paper clips

I I white porcelain pans

□

headlamps

u

current meter

□

large buckets

□

cinder blocks

□

planks

□

ruler

□

field recording sheets

u

toothbrush

□

small cup or scoop

u

styrofoam cooler

Dve

Studies

I I stop watch

I I rhodamine dye

107

Collecting and Sampling -pear (cont)

Water Quality Sampling

Miscellaneous Items

□
□
□
□
□
□

dipper

glass and/or plastic vials

glass and/or plastic bottles

DO bottles and rack

fixatives and preservatives

pH buffers

pH meter

field notebooks, pencil

tool kit

□
□
□
□

□

coolers, ice

USGS topographic maps

clipboard

field data sheets, maps

pencils, pens

waterproof markers

tags and labels (with

elastics or strings)
insect repellent

Chlorine Titrations

□

□
□
□
□
□

□
□
□
□
□
□

Fisher and Porter™
amperometric titrators
pH 4 buffer
5% KI solution
PAO tit rant
Q-tips™

Bon-Ami™1 cleaner
distilled water
200 ml beakers
1000 ml volumetric flask
1 ml pipets
Chlorox™ bleach
miscellaneous fuses

□

□
Zl
□

□

□
□

sunscreen

camera

film

paper towels

hand lens

disposable gloves

first aid kit

lab goggles

108

CAGED MINNOW TOXICITY ASSESSMENTS

3.6.5 DATA RECORD SHEET

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110

CAGED MINNOW TOXICITY ASSESSMENTS

3.6.6 REFERENCES

1. Rand, M.C., A.E. Greenberg, M.J. Taras, and M.H. Franson, (eds.). 1975.
Standard Methods for the Examination of Water and Wastewater, 14th edition.
American Public Health Association, Washington, D.C. xxxix + 1193 p.

2. O'Brien, W.J. and D. Kettle. 1981. A Zooplankton Bioassay Chamber for Lab
and Field Use. Journal of Plankton Research. 3(4): 561-566.

3. Jones, P. A., PH.D. (ed. ) . 1985. New York State Manual for Toxicity Testing
of Industrial and Municipal Effluents. Division of Water. New York State
Department of Environmental Conservation, Albany, New York,
(miscellaneously paginated)

4. Peltier, W. 1978. Methods for Measuring the Acute Toxicity of Effluents
to Aguatic Organisms. EPA-600/4-78-012. Environmental Monitoring and
Support Laboratory, Office of Research and Development, U.S. EPA,
Cincinnati, Ohio. 52 p.

5. Szal, G.M. , P.M. Nolan, L.E. Kennedy, C. Philbrick Barr, and M.D. Bilger.
(in preparation). Instream and Laboratory Evaluations of the Toxicity of
Chlorinated Wastewaters. Massachusetts Department of Environmental
Protection, Westborough and United States Environmental Protection Agency,
Lexington.

6. Technical Services Branch. 1989. Basins Program Standard Operating
Procedures River and Stream Monitoring. Massachusetts Division of Water
Pollution Control, Department of Enviromental Quality Engineering,
Westborough. v + 52 p.

Ill

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.7 Microtox™ Analysis 113

3.7.1 Definition 113

3.7.2 Objectives 113

3.7.3 Field Sampling 113
Sample Container Preparation 113
Sample Collection and Handling 114

3.7.4 Laboratory Analysis 115
Laboratory Equipment and Related Supplies 115

3.7.5 Quality Assurance 115

3.7.6 Field Equipment and Supply List 116

3.7.7 Interpretation and Reporting of Microtox™ Results 117
Test Description 117
Data Interpretations 117
Microtox™ Results Reporting Form 118

3.7.8 Microtox™ Sediment Toxicity Testing 119
Laboratory Equipment and Related Supplies 119

3.7.9 References 120

112

MICROTOX1

3.7 MICROTOX™ ANALYSIS

3.7.1 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.

3.7.2 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 .

3.7.3 FIELD SAMPLING

Both grab and composite samples can be analyzed for acute toxicity with
the Microtox™ Toxicity Analyzer. Generally, grab samples are taken at
instream stations while composite samples of effluents are collected at
municipal and industrial wastewater treatment facilities. Although the
compositing technique does have an "averaging" effect (which might mask
a peak of toxicity), it appears to be a good indicator of average
toxicity conditions. Composite samples are collected by ISCO automatic
samplers .

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.

113

Sample Collection and Handling

The following procedure is used to collect samples for Microtox™ analy-
sis :

1. Clean 16-ounce glass jars with teflon-lined caps should be filled
completely to eliminate any headspace.

2. Samples can either be grabs or composites but must "match" samples
sent for chemical analysis.

3. Both pH and Total Residual Chlorine (TRC) readings should be taken
in the field and noted on the sample tag. If the sample is a com-
posite then the TRC measurement should be taken on the composite.

4. The following parameters should be analyzed to the detection limit
(mg/1) specified:

Total Hardness 0.5

Total Alkalinity 2.0

TRC 0.02

Ammonia-Nitrogen 0.1

TKN 0 . 03

TOC 0.5

Specific Conductance —
Total Suspended Solids
Total Metals:

Ag, Cd, Cu, Pb 0.005

Cr, Ni 0.1

Al, Fe, Zn 0.2

5. The samples should be stored on ice for transport back to the
laboratory.

Sampling information noted on the sample tags include:

Source/Town:

Sampling Dates : (Both dates if 24h composite)

Sample Type: (Grab or composite)

Sampling Location: (Exact location/description)

p_H:

TRC:

Upon arrival at the Microtox™ Laboratory each sample is assigned a
log number and corresponding sample information is recorded in the
Microtox™ notebook. Before the sample is analyzed, both pH and TRC
are tested in the Microtox™ Laboratory and the results recorded. If
necessary, the sample is dechlorinated with sodium thiosulfate prior
to the toxicity analysis as chlorine is very toxic to the Microtox™
bacteria. The maximum holding time for a sample after collection is
24-hours.

114

MICROTOX™

3.7.4 LABORATORY ANALYSIS

The basic procedure for the Microtox™ system employs duplicates of both
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 1 pipet, micropipette tips 1-100 1

10. Eppendorf 500 1 pipet, micropipette tips 101-1000 1

11. parafilm, kimwipes

12. disposable gloves

13. Hach Kit Model DR100 Colorimeter

3.7.5 QUALITY ASSURANCE

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

115

3.7.6 FIELD EQUIPMENT AND SUPPLY LIST

Collecting and Sampling Gear

I 450 ml borosilicate type glass containers with caps
[ Orion model 201 field pH meter
[ rubber gloves

Miscellaneous Items

tags, labels, elastics
[ pencils , pens
[ plastic wrap

first aid kit
| cooler, ice

116

MICROTOX1

3.7.7 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
EC50 to toxicity is an inverse one; i.e., the lower the EC50J the greater the
toxicity of the sample.

A useful conversion of the EC50 is the Toxic Unit. This is simply the inverse
of the EC50 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 (%) 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 ECj^o (sample 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.

117

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

EC2o

EC50

TOXIC
UNITS
(T.U.)

NOTE: RESULTS GIVEN AS % VOLUME OF SAMPLE

118

MICROTOXm

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 EC50 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

3.7.8 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 Land 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

119

3.7.9 REFERENCES

1. Beckman, Inc. 1980. Microtox1" 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
Microtox1" Laboratory and Presentation of Several Case Studies Using
Microtox1" 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.

120

CHLOROPHYLL

SECTION PAGE

3.0 BIOLOGICAL FIELD AND LABORATORY METHODS

3.8 Chlorophyll Analysis 122

3.8.1 Definition 122

3.8.2 Equipment Needs 122

3.8.3 Log-In Procedure 123

3.8.4 Sample Preparation 123

3.8.5 Analytical Procedure 124
Calculation of Chlorophyll Concentrations 125

3.8.6 Instrument Calibration 126

3.8.7 References 127

121

3.8 CHLOROPHYLL ANALYSIS

3.8.1 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 .

3.8.2 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

122

CHLOROPHYLL

3.8.3 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.

3.8.4 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.

123

3.8.5 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 HC1 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.

124

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 is used.

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 mg/m^.

125

3.8.6 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.

126

CHLOROPHYLL
3.8.7 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.

127

4.0 QUALITY ASSURANCE

128

QUALITY ASSURANCE

SECTION PAGE

4.0 QUALITY ASSURANCE

4.1 Purpose and Scope 130

4.2 Intralaboratory Quality Assurance 130

4.3 Interlaboratory Quality Assurance 131

4.4 References 132

129

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
biomonitoring 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.

130

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
taxonomists' responses.

131

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).
1985. 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.

132

LABORATORY AND SAFETY CONCERNS

5.0 LABORATORY AND SAFETY CONCERNS

133

SECTION PAGE

5.0 LABORATORY AND SAFETY CONCERNS

5.1 Equipment
Personnel
Lab Equipment

5.2 General Laboratory Concerns
Ventilation
Handling of Chemicals

5.3 General Laboratory Clean-up Procedures

5.4 Handling Hazardous Materials
Spills

How to Use the Acid or Alkali Spill Kits
Hazardous Waste Storage

5.5 Laboratory Information by Task
Chlorophyll a Extraction
Macroinvertebrate Sorting
Fish Preservation
Toxicity Testing

5.6 References

135

135

135

135

135

135

135

135

136

136

136

136

136

136

137

137

137

138

134

LABORATORY AND SAFETY CONCERNS

5.0 LABORATORY AND SAFETY CONCERNS

5.1 EQUIPMENT

Personnel

Masks - for organic vapors

Face Shields

Goggles

Gloves

Aprons

Lab Equipment

Fume-hood

Spill kits

Fire extinguisher

First-aid kit

Chemical waste containers

Eye Wash Stations

5.2 GENERAL LABORATORY CONCERNS

Ventilation

The TSB laboratory, which is located in the basement of the Westview
Building, is equipped with a non-vented fume-hood. This fume-hood is
not vented to the outside; instead, it has a filter containing activated
charcoal which has a certain fixed capacity to absorb fumes from acids
and solvents. To be used correctly, the front door should be down and
the blower should be turned on. The fume-hood's blower and light should
always be shut off after use. Chemicals should not be stored under the
hood.

Handling of Chemicals .

If wash bottles are used to dispense chemicals, separate the inner
"pipe", located under the cap, from the upper portion before storing.
This will stop chemicals from dripping out while stored. Solvents, such
as acetone are extremely flammable and explosive. Care should be used
when transferring solvents and other related materials from container to
container.

5.3 GENERAL LABORATORY CLEAN-UP PROCEDURES

1) Keep bench tops and all work areas clear.

2) Wipe down all bench tops after use with a sponge or wetted paper
towels .

3) All glassware must be washed with a phosphorus-free soap (such as
Alconox") and rinsed twice with distilled water.

135

4) Bottles used for algae samples should be washed with Alconox™; rinsed
twice and then washed with 10% hydrochloric acid (HCI) followed by
three rinses with distilled water.

5.4 HANDLING HAZARDOUS MATERIALS

Spills

Quickly rinse off in cool water any chemical to which skin is exposed.
Acids should be flushed from the skin for at least 15 minutes. Spill
kits for acids and alkali chemicals are located below the chemical waste
storage table.

How to Use the Acid or Alkali Spill Kits

1) Contain the spill by distributing absorbent around the perimeter.

2) Sprinkle the acid or alkali neutralizer beginning at the perimeter
and working inwards.

3) When the reactions begin to slow, begin mixing with the scoops
provided to insure that all the acid/alkali has been neutralized.

4) Use plastic scoops to gather up the absorbent and neutralizer and
transfer to plastic bags.

5) Wipe up areas with a damp sponge.

Hazardous Waste Storage

Any chemical wastes that are generated should be stored in labeled glass
containers. These should be kept on the table in the laboratory which
is labeled Chemical Waste Storage until full. Labels should be affixed
to the bottle rather than attached with a rubber band. Transfer of
waste material into a similarly labeled waste can (i.e., acids,
solvents, bases) should be done if proper waste cans are available. If
waste cans are not available then the labeled waste containers should be
stored in the barrels provided.

5.5 LABORATORY INFORMATION BY TASK

Chlorophyll a Extraction

Avoid breathing acetone fumes as they can irritate the lining of the
nose and throat. Avoid contact with skin; prolonged exposure can result
in skin rashes. Different grades of acetone are used for different
tasks. For chlorophyll a analysis use only those bottles marked "For
chlorophyll a analysis only." Gloves and goggles, should be used when
working with acetone. The area should be well ventilated; to ensure
this, use the fan when necessary, or face masks which filter organic
vapors .

136

LABORATORY AND SAFETY CONCERNS

Bottles which have held the algae/chlorophyll a samples should be acid
washed before being used again. To make the 10% HCI solution, add 10 mis
of concentrated hydrochloric acid to 90 ml of distilled water. Always
add acid to water and not the reverse. Remember the slogan: "Add acid
to water just like you oughta." Gloves, goggles and aprons should be
used when working with concentrated acids.

Acetone wastes should be stored in clearly labeled bottles; do not store
with nitric or sulfuric acid wastes.

Macroinvertebrate Sorting

Ethyl alcohol is used to preserve samples. Gloves and goggles should be
used when handling alcohol. When samples are brought back from the
field they are in the highest concentration of alcohol (95%). It is
particularly important at this stage to work in a well ventilated area -
usually by the sink in the lab. Alcohol is flammable; if a fire does
occur in the lab use the fire extinguisher located in the hallway. Do
not use water to put out the fire.

CMC Mounting medium, used for making microscope slide preparations,
carries the following warning:

"[This is] a chemical in concentrated form. Avoid contact with skin or
eyes. Do not take internally - may be fatal if swallowed. In case of
contact with eyes flush with water for at least 15 minutes. If
swallowed, and for eye contact, obtain medical attention immediately."

Fish Preservation

Fish for long-term preservation are fixed in 10% formalin before they
are transferred to 50% isopropanol. Both formalin and isopropanol are
irritants. Formalin, especially, can irritate the respiratory tract.
Care should be taken when halding either of these chemicals. Use the
fume-hood and/or face masks when making dilutions or transfering
materials .

Toxicity Testing

Phenyl arsene oxide is used for chlorine titrations using the ampero-
metric titrators. In high concentrations it is very toxic. Care must
be taken to avoid inhalation of this compound. Do all titrations under
the hood. Label waste containers clearly and store separately from
solvents .

Unchlorinated wastewater samples are often analyzed using the Microtox"*.
Gloves should be worn when handling the sample containers which have
often been in contact with the sample effluent, as well as when working
with the sample. Gloves, pipettes, vials which have been in contact
with fecal material should be promptly disposed of in a waste receptacle
outside of the building.

137

5.6 REFERENCES

1. Bewick Associates, Inc. 1984. Employer's Right-To-Know Manual: A
Step-by-Step Guide to Implementing Massachusetts Right-To-Know Law. Bewick
Associates, Newton, MA. iv + 10 sections (unpaginated) .

2. Steere, N.V., (ed.). 1986. CRC Handbook of Laboratory Safety. CRC Press,
Inc., Boca Raton, Florida . xiii + 854 p.

138

GENERAL BIOLOGICAL FIELD AND LABORATORY REFERENCES

6.0 GENERAL BIOLOGICAL FIELD AND LABORATORY REFERENCES

139

6.0 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.

140

GENERAL BIOLOGICAL FIELD AND LABORATORY REFERENCES

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.

141

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
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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.

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