Ecological Research Series
GUIDELINES FOR ZOOPLANKTON SAMPLING
IN QUANTITATIVE BASELINE
AND MONITORING PROGRAMS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-78-'
February 1978
GUIDELINES FOR ZOOPLANKTON SAMPLING IN
QUANTITATIVE BASELINE AND MONITORING PROGRAMS1/
by
Fred Jacobs and George C. Grant
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062
Grant Number -
EPA-R804147010
Project Officer
Richard C. Swartz
Newport Field Station
Marine and Freshwater Ecology Branch
Corvallis Environmental Research Laboratory
Newport, Oregon 97365
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON. 27330
Special Scientific Report No. 83, Virginia Institute of Marine Science
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental Research Lab-
oratory, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or recommen-
dation for use.
ii
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Protection
Agency would be virtually impossible without sound scientific data on pollu-
tants and their impact on environmental stability and human health. Respon-
sibility for building this data base has been assigned to EPA's Office of
Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory (CERL).
The primary mission of the Corvallis Laboratory is research on the effects
of environmental pollutants on terrestrial, freshwater, and marine ecosystems;
the behavior, effects and control of pollutants in lake systems; and the
development of predictive models on the movement of pollutants in the
biosphere.
This report presents a review of methods for sampling and analyzing marine
zooplankton communities. These quantitative techniques can be used to
establish ecological baselines or to conduct surveys of the impact of
pollution on zooplankton dynamics.
A. F. Bartsch
Director, CERL
111
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ABSTRACT
Methods applicable to zooplankton sampling and analysis in quantitative
baseline and monitoring surveys are evaluated and summarized. Specific
recommendations by managers must take into account characteristics of the
water mass under investigation, the abundance of contained zooplankton and
phytoplankton populations and the objectives of the study. Realistic
planning and development must also consider available monetary and manpower
resources.
This report was submitted in fulfillment of Contract No. R804147010 by the
Virginia Institute of Marine Science under the sponsorship of the U. S.
Environmental Protection Agency. This report covers a period from 24 Nov
75 to 31 May 77, and work was completed as of 25 Feb 77.
iv
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CONTENTS
Foreword
Abstract
Contents
Acknowledgments
Sections
1 Conclusions
Introduction 3
2.1 Zooplankton 3
2.2 Spatial variations 3
2.3 Temporal variations 4
2.4 Introduction to bias and variation associated with 4
sampling
2.4.1 Introduction to sampling gear bias 4
2.4.2 Avoidance of samplers by zooplankton 5
Sampling 6
3.1 Sampling site selection and station selection 6
3.2 Sampling gear 7
3.2.1 Water bottles 7
3.2.2 Pumping systems 7
3.2.3 Introduction to net sampling 8
3.2.4 General considerations of net sampling 8
3.2.5 Recommended nets for oceanic sampling 10
3.2.6 Care of nets 10
Shipboard handling of samples 12
4.1 Labelling and shipboard handling of samples 12
4.2 Preservation of samples 12
4.3 Shipboard procedures for obtaining samples 14
4.4 Special cases . 14
4.4.1 Sampling for pollutants 14
4.4.2 Sampling gelatinous organisms 15
Sample processing 16
5.1 Subsampling by pipette method 16
v
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Sections
Sample processing (cont.)
5.2 Subsampling by splitting 16
5.3 Biomass 17
5.3.1 Introduction 17
5.3.2 Shipboard treatment 18
5.3.3 Settling volume 18
5.3.4 Displacement volume 18
5.3.5 Wet weight 19
5.3.6 Dry weight 20
5.3.7 Ash-free dry weight 20
5.3.8 Large gelatinous forms 20
Additional biochemical analyses 21
6.1 Introduction 21
6.2 Total protein 21
6.2.1 Biuret method 21
6.2.2 Lowry (et al.) method 21
6.3 Total lipid analysis 22
6.4 Carbohydrate 22
6.5 Ash 22
6.6 Chitin 23
6.7 Other analyses 23
6.7.1 Amino acids 23
6.7.2 Fatty acid analyses 23
6.8 Biochemistry of marine zooplankton 24
Data Analysis 25
7.1 Total numbers and frequency of occurrence 25
7.2 Diversity indices 25
7.3 Multivariate analysis 27
Sectional bibliography 28
VI
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ACKNOWLEDGMENTS
The authors wish to thank Dr. Richard C. Swartz of the Environmental Protec-
tion Agency for his helpful suggestions in developing this report and for
critically reviewing the final draft. We also thank Mrs. Shirley Sterling
for her very able secretarial assistance.
vii
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SECTION 1
CONCLUSIONS
The zooplankton is a diverse assemblage of animal forms, and as a result,
exhibits wide spatial, diurnal and seasonal variations in abundance and
composition. Accurate interpretation of quantitative and qualitative
relationships are further complicated by gear bias and the ability of
certain groups to avoid capture. Methods to minimize error estimates
follow no standard procedure; only careful analysis of the problem under
study facilitates the proper approach.
Baseline studies are required to be broader in scope and more detailed in
their enactment than are monitoring studies. Frequency of sampling, number
of stations, accumulation of hydrographic information, etc., should be
expansive for baseline studies. Monitoring should be initiated when phe-
nomena are somewhat understood and/or patterns from previous baseline
studies have been developed. As a result, stations in monitoring studies
are usually set further apart than in original baseline studies. However,
characteristics of the water column being sampled are most important in
determining the location of sampling sites. Sampling locations might be
further apart in homogeneous waters, such as certain offshore areas, than
in heterogeneous coastal waters. In estuaries, where environmental para-
meters exhibit considerable variations over relatively short distances and
time periods, a closer spacing of sampling sites and an increased frequency
of sampling are recommended. Sampling sites are often randomly selected
stratified stations chosen from a gridded pattern which has been overlaid
on the study area. Transects may be utilized in situations where the study
area covers great distances and ship time is limited. In pollution studies,
a series of transect lines radiating from a source point may be advisable.
Furthermore, stations along these transects should extend into unaffected
areas. In studies concerned with small scale distribution of zooplankton,
a parachute drogue can be employed to maintain station locations for repeated
sampling from the same water parcel.
Pumping systems, although expensive, are most efficient for capturing micro-
zooplankton, but these systems should pump in excess of 150 1/min. to min-
imize avoidance.
Nets are recommended for sampling mesozooplankton. There is no ideal
plankton net; appropriate gear selection is dictated by a clear understanding
of the problem being studied. The mouth opening of the net should be as
large as possible, and still retain its ability to be handled efficiently
aboard ship. Nets with mouth openings of 50 to 100 cm in diameter (or 0.2 m2
or greater) are adequate for capturing most groups. Tow speed should be
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between 1 1/2 - 2 knots for low speed nets, and the net should maintain a
constant velocity while under tow. Mesh size selection is critical in deter-
mining quality and quantity of the catch. In relatively plankton rich,
temperate coastal waters, 333 ym is the minimum mesh size that maintains a
filtration efficiency of 85% or greater. Although a 202 ym mesh net has the
advantage of retaining forms that pass through a 333 ym mesh net, filtration
efficiency drops after 5 minutes of tow in these waters. In areas of low
plankton biomass, where clogging is not likely to occur, the 202 ym net is
recommended. All nets should be properly cared for, kept out of direct sun-
light, and washed in fresh water at the conclusion of each cruise.
Samples should be properly labelled and generally are best preserved in 4%
buffered formaldehyde. Sampling zooplankton for pollutants requires special
methodology, largely designed to minimize potential contamination. Splitting
of samples is recommended for quantitative studies; two types of splitters
are suggested, the Folsom & the Burrell. Zooplankton groups should be split
to workable numbers (usually 100-200 individuals within a group).
Paired nets provide a replicate sample that can be utilized for biomass
considerations. The most popular techniques for presenting biomass are:
(1) Settling volume, (2) Displacement volumes, (3) Wet weight, (4) Dry weight
and (5) Ash-free dry weight. Volume measurements and wet weight are non-
destructive measurements. Information on the nutritional content of the
zooplankton is provided by dry weight and ash-free dry weight. Dry weight
is best determined by freeze-drying samples.
The patchy distribution of zooplankton in the marine environment dictates
care in the application of appropriate statistical methods of analysis.
Observations made from ranking, abundances of individual groups, species
and biomass, still form the bulk of the accumulated data from baseline and
monitoring surveys. Spatial and seasonal trends can be interpreted in this
manner. Diversity indices can be used for zooplankton community comparisons
and pollution studies. These indices may have added value when used in
conjunction with other indices, associated statistics and observations.
With the widespread availability of computers, multivariate approaches are
receiving greater attention. This type of analysis draws its conclusions
by recognizing patterns among variables and by condensing multidimensional
relationships. By using clustering techniques, samples may be partitioned,
distinguished and ranked, according to similarities of species composition
and abundance.
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SECTION 2
INTRODUCTION
2.1 Zooplankton
The term zooplankton refers to individuals and communities of animals, whose
distribution and dispersal are influenced significantly by the movements of
the waters. Zooplankton are essential intermediary links in marine food
chains since they graze on phytoplankton, and provide a direct food source
for more complex animal forms. Furthermore, the zooplankton impart excretion
products and organic detritus to the marine environment, and planktonic
organisms constitute a considerable portion of the biomass of the world's
oceans. Almost every animal phylum is represented, at least at some life
stage, in the plankton.
This great diversity of animal types makes the zooplankton a difficult com-
munity to work with. Specialists are required for individual groups and
quantitative sampling is beset with problems. In addition to errors asso-
ciated with present sampling methodology, a greater source of sampling
difficulty is the "aggregate" or "patchy" nature of the zooplankton itself.
2.2 Spatial variations
In the late 1800's and early 1900's opposing opinions concerning the distri-
bution of oceanic plankton were held. Despite Haeckel's (1890) efforts to
persuade the scientific community that, "the composition of the plankton is
in qualitative as well as quantitative relations, very irregular," Victor
Hensen, working with Kiel planktologists, firmly advocated relatively even
distribution. Hensen's theory implied that plankton data obtained from a
few hauls, could be used to estimate conditions over a much greater area of
the ocean. Haeckel's hypothesis implied that individual areas of the sea had
to be sampled before anything could be stated about plankton communities
within these areas. The controversy was ended by results of A. C. Hardy's
(1936) "Discovery" and "William Scoresby" expeditions. By utilizing a con-
tinuous plankton recorder, which sampled plankton densities and fluctuations
along continuous lines of observation, Hardy concluded that "there is no
doubt as to the patchy nature of the oceanic plankton."
Explanations offered for the existing patchy distribution of oceanic plankton
are numerous, ranging in physical, chemical and biological factors. Selected
references dealing with this subject are presented in the bibliography on
this section. These aggregates of zooplankton vary in size and shape, but
several researchers have indicated patch diameter to be hundreds of meters
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or less. Densities within patches are generally from 2 to 5 times greater
than background densities. Results from computer simulated models and actual
field studies have indicated that the size and distribution of patches in a
body of water greatly affect the accuracy and precision of estimates of
zooplankton abundance.
2.3 Temporal variations
Natural fluctuations associated with seasonal variations in sunlight inten-
sity, water temperature and transparency, oxygen and nutrient content of the
water etc. exert direct and indirect effects on zooplankton survival and
community development. As a result, wide ranges in zooplankton biomass and
specific abundance occur. At a single station in the Sargasso Sea, variations
in biomass changed by a factor of 10, between certain seasons (Deevey 1971).
In Chesapeake Bay, biomass in summer months can be two orders of magnitude
greater than in relatively sparse periods; differences in actual numbers of
individuals within groups can vary by as much as 5 or 6 orders of magnitude
between seasons (Jacobs and Grant MS.). Furthermore the change of dominant
species, over time, results in distinctive zooplankton communities during
certain periods of the year. The degree to which naturally occurring seasonal
phenomena affects variability in zooplankton numbers and species must be
considered when designing a zooplankton study.
Many zooplanktonic organisms perform diurnal vertical migrations, the effects
of which are most pronounced in the upper 300 m. An early model indicated
that upward migration begins at noon, and organisms attain their highest water
column levels near midnight. The effects of light and the physiology of the
organisms themselves appear to be the most important controlling factors in
diurnal migration. The likelihood of sampling error is further increased
during darkness since migrating species tend to be compressed into more
discrete layers during night hours, than are nonmigrating species. An
important consequence of vertical migration is that no two samples from
the same body of water, unless taken at the same time of day, are directly
comparable. In "Plankton and Productivity of the Oceans", Raymont (1963)
devotes a chapter to vertical distribution of zooplankton and reviews the
early literature on diurnal migration.
There appears to be, at least in nearshore waters, a periodic fluctuation in
zooplankton abundance and composition that is directly related to tidal con-
dition. This is generally more prominent in species that do not undergo diel
vertical migrations (Sameoto 1975).
2.4 Introduction to bias and variation associated with sampling
2.4.1 Introduction to sampling gear bias
In addition to variations related to the plankton and its environment, addi-
tional error and bias is introduced by the choice of gear, and the manner in
which it is used. Proper gear selection is essential in attempting to max-
imize accuracy, but no "blanket" recommendations for the "ideal" gear types
can be offered. Only careful analysis of the problem under study will
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facilitate the correct choice(s). Naturally, factors such as ship time and
capability, funds for study, and available manpower are, practically speaking,
as important as the questions under study, for selecting gear. With many
samplers, variables such as the speed, depth and duration of tows can affect
resulting catches. When nets are used, different mouth openings, mesh sizes,
netting material and net structure influence the biomass and constituency of
the captured organisms. This report will present a variety of sampling sit-
uations and offer appropriate suggestions and recommendations to maximize
sampling efficiency.
2.4.2 Avoidance of samplers by zooplankton
Although discussed in greater detail later in this report, the subject of
avoidance deserves brief mention here. Error and variation in estimating
zooplankton is increased by the ability of certain groups to avoid capture
while others cannot. This results in selective sampling and in general
underestimation of zooplankton abundance and biomass. Mechanisms of avoidance
are numerous: responses to changes in water movements, size, swimming abil-
ity, etc. of the animals are all factors. Certain age or physiologically
stronger groups within a species may exhibit a greater ability to avoid
capture than others. Naturally, avoidance is greatly influenced by the
type(s) of gear utilized, and the manner in which it is used. For further
information, a UNESCO report that summarizes the exhaustive literature on
avoidance, is recommended (Clutter and Anraku 1968).
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SECTION 3
SAMPLING
3.1 Sampling site selection and station selection
The problem under study, time, and available money, are essential consider-
ations for sampling site selection. The type of information required dictates
the scientific approach. For example, baseline and monitoring studies,
although related, have distinct differences in the scope of information they
seek, and therefore require different thought processes in their development.
Subsequently, the location, frequency, and number of stations sampled should
be quite comprehensive for initial baseline studies. Baseline studies should
also include intensive sampling of a wide variety of related measurements,
such as dissolved oxygen, salinity, temperature and other aspects of water
chemistry (i.e. nutrients). Selection of sampling sites can be facilitated
by dividing the area into a gridded pattern or into transects. Once data
and information are analyzed, phenomena are better understood and trends
established, scaled down monitoring can be initiated. Numbers of stations
and samples can be reduced, if it is feasible to do so without information
loss. Essentially, monitoring programs should be designed to provide maximum
information for minimum cost and effort.
The design of the sampling program must consider both the objectives of the
investigations and the hydrographic conditions in the area of study. Sampling
locations might be further apart in offshore areas of homogeneous water char-
acteristics, than in rather heterogeneous neritic areas, where close sampling
sites might be indicated. In estuaries, where environmental variables exhibit
considerable variations over relatively short distances, an even closer
spacing of sampling sites is recommended.
In oceanic studies, the sampling patterns may consist of a series of transects
across different current systems, a series from shore to deeper waters, or a
grid of nearly equidistant stations within an oceanographic area. Although
gridded mapping of a study area is most desirable, this is not always possible
in studies that monitor large distances and have fixed time requirements.
When a grid pattern is utilized, it is usually overlaid on a study area that
has been divided into subareas. These subareas are generally determined by
hydrographic factors. Within each subarea, random stratified stations may be
selected. It is advisable to draw up a cruise track having a clear indication
of the projected ship time, both available and required, to complete the
station program. It is recommended that additional alternate stations are
included in the sampling program. Furthermore, researchers should pre-deter-
mine which stations are "least crucial" to the study, in the event that time
factors dictate elimination of certain stations.
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Frequency of sampling is also dependent on the specific objectives to be
answered and available ship time. Generally, ship requirements differ for
different studies; estuarine areas can, as a result, be more frequently
sampled than offshore waters. It is desirable and feasible to conduct
surveys in estuarine waters monthly or biweekly, while offshore monitoring
is usually conducted bi-monthly, quarterly or semi-annually. All available
published literature and hydrographic data accumulated for an area of poten-
tial study should be utilized to maximize efficiency of sampling design.
In cases of pollution related monitoring problems, it is preferable that a
series of samples should extend from affected to unaffected areas, the latter
samples then serving as controls. If this is not possible, control data can
be provided from surveys in the same area, conducted previous to the time of
the introduction of a pollutant, or from other unpolluted areas of similar
hydrographic and environmental structure. Sampling sites should be close
enough to show gradient effects when they exist. Monitoring should continue
until recovery is complete. In studies that monitor effects from a direct
environmental addition, stations can be established along a series of
transect lines, radiating from a source point.
For sampling a specific water parcel, the use of a parachute drogue, set below
the surface, provides a suitable reference point for navigation and station
location. Relative positions of station sites are maintained, since modifi-
cations in the sampling locations are determined by movements of the drogue.
In this way the same parcel of water can be repeatedly sampled, regardless of
the influence of the currents. This is particularly useful in field studies
that are concerned with small scale variations in zooplankton.
3.2 Sampling gear
Three general classes of sampling gear are used to sample zooplankton, namely
water bottles, pumps, and nets. This section will devote a major emphasis on
net samplers, since they are most efficient for capturing organisms larger
than micro-zooplankton.
3.2.1 Water bottles
The use of water bottles provides samples and associated hydrographic data
from fixed locations within the water column. For most purposes 10 liter
bottles are recommended. These samplers can capture and return live organ-
isms; however, animals capable of only moderate locomotion can avoid capture.
Water bottles, widely used in phytoplankton sampling, are of value only in
sampling microzooplankton (animal plankters that pass through a 202 pm mesh
net) .
3.2.2 Pumping systems
This is probably the most favorable method for capturing microzooplankton.
These systems generally control filtering aboard ship, with water being run
through a single (or a nest of different sized) filter(s). Samples are then
preserved and generally analyzed by direct counts with an inverted microscope.
Pumping systems can be towed from moving vessels and can provide integrated
samples over a range of depth. The depth range capability is a function of
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the amount of hose attached to the pump. To minimize avoidance these systems
should pump in excess of 150 1/min. If the capture of larger forms (i.e.
copepods) is desired, pumping rate should be in excess of 200 1/min. In
certain cases, pumping may cause partial or total destruction of soft bodied
forms. Another disadvantage of pumping systems is that they tend to be
expensive and require specific shipboard modifications.
3.2.3 Introduction to net sampling
Although this is the best method for capturing larger forms, sampling micro-
zooplankton with nets is not recommended. To retain microzooplankton, net
meshes must be small and as a result, samplers tend to clog rapidly. A 103
urn mesh has been demonstrated to fall below 85% filtering efficiency during
the first minute of tow (Smith et al. 1968).
The grouping of zooplankton by size classes generally follows this scheme:
(1) microzooplankton - defined as zooplankton that pass through a mesh
size of 202 ym;
(2) small mesozooplankton - defined as zooplankton retained by a mesh size
of 202 ym;
(3) large mesozooplankton - defined as zooplankton retained by 1 mm mesh
size;
(4) macrozooplankton - defined as large agile plankters and less agile
nekton (captured by such gear as an Isaacs-Kidd Midwater Trawl).
Although there is no "ideal" zooplankton net available, attempts should be
made to limit choices for given situations. This would improve standard-
ization and result in greater comparability of different studies. Re-
searchers, however must have a clear definition of the problem under study;
this generally facilitates the appropriate choice of gear. For example,
Vannucci (1968) pointed out the differences in approach when sampling for
fish larvae and mixed plankton. "The fisheries manager tries to select
certain size classes and or species against the others, while the plank-
tologist tries to obtain as representative a sample as possible of the
mixed association living in the sea." A 505 pm mesh net may be adequate
for sampling fish larvae, but would be of limited scientific value for
sampling immature copepods and other smaller forms.
3.2.4 General considerations of net sampling
(1) Mouth opening - nets with different sized mouth openings have been shown
to have selective capabilities for the capture of zooplankton spe-
cies. The importance of mouth opening for the efficiency of capture has
been examined in actual field and computer simulated models. It appears
that 20 and 40 cm nets introduce error by underestimating abundance and
diversity (McGowan and Fraundorf 1966; Wiebe and Holland 1968). Larger
mouth openings (100 cm or greater) yield more precise data and capture
larger forms. However the larger the net, the more difficult it is to
handle and retrieve. Although larger nets are more desirable, nets with
mouth openings of 60 cm diameter (or 0.2 m^) are considered adequate for
ocean sampling. When sampling for macrozooplankton, little difference
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was noted between 6 and 10 foot Isaacs-Kidd midwater trawls for most
groups, excluding the fishes (Friedl 1971). The 10 ft trawl captured
significantly more numbers and individual species of fish, than did the
6 foot trawl.
(2) Tow speed - This can be an important factor, considering the avoidance
capabilities of certain species. Tested species of copepods were able
to avoid nets towed at 30 cm/sec (0.6 knots). It is therefore recom-
mended that low speed nets be towed between 1 1/2 and 2 knots (75-100
cm/sec). Even at constant engine speeds (between 1 and 2 knots),
variation in net speed through the water sometimes exists, and may
introduce significant error in the interpretation of data.
High speed samplers (>3 knots) have been developed for use from moving
vessels. Although records indicate that these samplers sometimes damage
organisms, other reports found these samplers to capture and return
intact organisms. Reduction of mouth opening size avoids conditions
of clogging in these nets. At speeds of 1 1/2-2 knots most plankton
are captured; high speed should be required only for sampling specific
groups (primarily fishes) capable of considerable movement.
(3) Mesh size - In temperate, relatively plankton rich coastal waters, it
has been demonstrated that 333 ym is the minimum net mesh size that
maintains over 85% filtration efficiency for the duration of 15 minute
tows (Smith et al. 1968). Smaller sizes tend to have reduced filtra-
tion efficiency due to clogging. Filtration efficiency is calculated by
comparing results of two flow-meters, one mounted inside the net mouth
opening, the other outside,
observed (from inside
Filtration Efficiency (%) volume of water through net flowmeter)
= theoretical maximum volume of water (from outside
through net flowmeter)
X 100
Since the 202 pm mesh net delineates microzooplankton from small meso-
zooplankton, it would appear logical that this net receive wide usage,
and it has. A UNESCO (1968) report recommended using a 200 \im mesh net
for sampling small mesozooplankton. Unfortunately, this net tends to
clog in plankton rich waters, filtration efficiency dropping below 85%
in 5 minutes. It does, however, have the advantage of retaining forms
that pass through 333 um mesh net. whether this would add significant
information to a problem under study, must be considered by individual
investigators. In areas where clogging is not likely to occur, this
mesh size (202 um) is recommended. ' -
(4) All nets should be made of nylon material.
(5) When nets are under tow, wire angle should be 30° or less. Nets should
not be raised faster than 45 meters/min during oblique or vertical tows.
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(6) Other factors such as filtration to mouth area ratios, bridles, color of
nets, extrusion through meshes, etc. can all affect the net's efficiency.
3.2.5 Recommended nets for oceanic sampling
Of the dozens of samplers available (see Jossi, 1970) only three types are
discussed below:
(1) Multiple opening-closing nets - These systems are now becoming popular
at certain major oceanographic laboratories. This system provides infor-
mation on intermediate (100's to 1000's of meters) scale spatial patterns
of zooplankton distribution and can be equipped with sensors that monitor
related environmental parameters. The sampler contains several nets,
each of which is opened and closed sequentially by commands through
conducting cable from the surface (Wiebe et al. 1976). This system
provides the best technology developed to date for studying the vertical
distribution and profiles of marine zooplankton species. This system
is, alas, quite expensive and large; its use is limited to well equipped,
large ocean research vessels.
(2) Paired opening-closing Bongo system - This sampler consists of two
mechanically operated opening-closing nets mounted side by side (McGowan
and Brown, 1966). These nets are recommended over single net samplers,
in that they provide two samples from the same environment. One can be
used for taxonomic purposes, while biochemical analyses can be conducted
on the other. The mouth opening of each net should be at least 50 cm in
diameter (60 cm is more desirable) and have a mesh area to mouth opening
area ratio of at least 5 to 1. Used concomitantly with a pinger and/or
a Time-Depth Recorder, this system yields an integrated sample over a
desired depth range. Digital flowmeters should always be used to monitor
water flow. This system is much less expensive than multiple opening-
closing nets and is easier to handle. Although multiple opening-closing
nets are preferable for studying detailed vertical structure, paired
opening closing systems are adequate when single integrated samples over
a depth range are required.
(3) Longhurst-Hardy Plankton Recorder - This sampler has been developed for
studying small scale patterns (10's to 100's of meters) of spatial zoo-
plankton distribution. This net utilizes the Hardy principle, of
capturing plankton on gauze in the cod end. The gauze is advanced at
periodic intervals, and can be changed by spooling. This sampler ex-
hibits a certain amount of bias, probably in part due to its relatively
low mesh aperture area to mouth opening area ratio (stalling and other
residence time biasing of organisms sometimes being significant). Re-
cently, Haury et al. (1976) have conducted several experiments on the
sources and degree of bias associated with Longhurst-Hardy Plankton
Recorders. Recommendations are made for proper design and use of this
sampler.
3.2.6 Care of nets
(1) After sample is removed from net, by carefully hosing organisms into cod
end, net is rinsed without a cod end.
(2) If clogging is severe, nets should be washed in detergent.
10
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(3) Net should never be left in direct sunlight for extended time periods.
(4) Both net and flowmeter should be washed in fresh water, after each
cruise (flowmeters washed more often during cruise, if possible).
11
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SECTION 4
SHIPBOARD HANDLING OF SAMPLES
Most of the recommendations from this section are taken directly from
Griffiths et al. (1976). Samplers should always be handled carefully in
a manner to minimize injury to zooplankters and scientists.
4.1 Labelling and shipboard handling of samples
Glass jars with screw on plastic lids are generally used for storing zoo-
plankton. Jars should be labelled on the outside with pre-printed gummed
labels, this can be done in the laboratory prior to ship time. Labels should
never be placed on jar caps. A sample label used by the Virginia Institute
of Marine Science (VIMS) Planktology Department is presented below:
VIMS PLANKTOLOGY DEPT.
SHIP CRUISE NO COLL. NO
DATE GEAR MESH SIZE
STA. NO. or LOCATION —.
In addition to outside labels, an internal label written on water-resistant
paper should be placed in every jar. Black India ink is preferred over pencil
marks because it provides a permanent, waterproof, easy-to-read record. In-
ternal labels should also be pre-printed.
Collecting information should be contained in« a field log as the samples are
being collected. A sample page from a VIMS field log is presented on the
following page. Generally, comments on sea state, hydrography and water
chemistry are kept on a separate form(s).
4.2 Preservation of samples
Formaldehyde is commonly used as a fixative for preserving zooplankton sam-
ples, a saturated solution containing 38-40% formaldehyde is known as,
"concentrated formalin". One part formalin should be added to nine parts
sea water, to provide a 4% formaldehyde solution. Buffering of concentrated
formaldehyde should be conducted by adding 2 gms. of borax to 98 ml of
12
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BLM Bongo Collections
Station No. Collection No.
Location (start) Depth
Location (end)
Date Vessel
Time (EST) opened Meter final rdg.
Time at depth " initial rdg.
Max fishing depth No. revs.
Time closed Meter No.
Net size and mesh Vessel speed
Type of tow (stepped or continuous oblique)
Depressor used Wire angle Wire out
(at max, fishing depth)
Remarks:
13
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concentrated formalin, before dilution. Periodically, the neutrality of the
sample should be checked with pH paper. Calcareous animals should be pre-
served in formalin with a pH of 8.2, since calcium carbonate dissolves below
that level. The use of instruments that can dispense formalin is recommended.
Furthermore, formaldehyde can be very harmful; it should be washed off skin
immediately. Splashes in eyes should be followed by washing in cold water for
10-15 minutes, and medical attention.
4.3 Shipboard procedures for obtaining samples
(1) Secure flowmeter(s) propeller (use of TSK or GO type meters are recom-
mended) . Read and record number of revolutions obtained during tow.
(2) Wash net from the outside, starting at the mouth and working downward
toward the cod end. Use a sea water hose whenever possible.
(3) Remove the bucket or bag from the cod end; concentrate sample through a
concentrating device of mesh size equal to or smaller than the mesh of
the net. This must be done quickly to minimize damage.
(4) Plankton should be washed into the labelled sample jar, filled 3/4 full
with sea water. The volume of the plankton should not exceed 10% of the
jar. If catches are larger, plankton should be placed in a larger jar
or divided into 2 jars. If divided, the jars should indicate either,
"1 of 2 samples" or "2 of 2 samples" etc.
(5) Add formaldehyde, preferably from a dispenser. Final concentration
should be 4%. Gently rotate jar.
(6) Place inside label in jar, seal cap tightly and invert the jar several
times periodically during the first hour of fixation.
(7) Before proceeding to next station, check to see that data have not been
omitted from field logs.
(8) Store the jars in a cool, dark, place.
(9) Upon return to the laboratory, the state of the sample preservation and
sample pH should be periodically checked.
(10) Shipboard procedures for obtaining biomass samples are discussed in
section 5.3.
4.4 Special cases
4.4.1 Sampling for pollutants
Problems arise when sampling zooplankton for pollutants, such as trace metals,
pesticides or petroleum hydrocarbons. Since these levels are extremely low
in the natural environment, and ocean-going vessels are a generous source of
these materials, the potential for obtaining contaminated specimens is great.
14
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Certain precautions can, however, be taken to minimize contamination:
(1) Specimens should never have any contact with the surface of the ship.
(2) Contact between collecting gear and ship surfaces should be minimal.
(3) Neither sample nor gear should be washed with the ship's salt water.
(4) Hands, tools, sorting trays and storage containers should be liberally
washed with 95% ethanol.
(5) Handling and sorting gear must be of metal, glass or enamel.
(6) Use metal cups for pesticide collections.
(7) Use of opening-closing nets can be used to avoid contamination from the
ship (i.e. paint chips). Sampler is opened and closed below the surface.
4.4.2 Sampling gelatinous organisms
Sampling gelatinous zooplankton with nets may prove to be inadequate for
certain groups. Many of these organisms are delicate and are lost or damaged
beyond recognition when strained through meshes. Furthermore, many of these
groups do not preserve well in formaldehyde and require specialized methodol-
ogy for fixation and preservation. Current information on biology and distri-
bution of these forms is being contributed from in situ observations made by
SCUBA divers.
Ctenophores
These organisms may appear in excessive numbers in plankton tows, especially
in temperate estuaries during certain times of the year. Since they tend to
break apart and (when dominating samples) somewhat gum up formaldehyde pre-
served samples, ctenophores should be removed. Their type, number and volume
should be recorded and they can be preserved separately. A reference for
fixing and preserving ctenophores is listed in the bibliography (Adams et al.,
1976).
15
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SECTION 5
SAMPLE PROCESSING
Once they are returned to the laboratory, processing of preserved samples can
be initiated. Before proceeding further, large organisms (i.e. medusae, fish
larvae, etc.), especially when uncommon to the sample, should be removed.
These forms are then counted and identified.
5.1 Subsampling by pipette method
A summary of this method is taken largely from an earlier U. S. Environmental
Protection Agency manual (Weber, 1973) and proceeds as follows:
(1) The sample is drained of excess formaldehyde; settled volume is read in
a graduated cylinder or Imhoff cone in a volume of water such that plank-
ton makes up 1/5 of the diluted volume.
(2) Sample is stirred with a Stempel pipette; 1 ml of the agitated mixture
is withdrawn from the sample.
(3) The subsample is transferred to a gridded culture dish with 5 mm squares.
(4) Pipette is rinsed with distilled water into culture dish, to remove any
adherent organisms. Enumerate and identify subsample under a dissecting
microscope.
After calculating the dilution factor, the information derived from counting
together with estimates of water volume sampled, can be used to derive the
total abundance of individual groups or of the total sample. However, con-
sidering the relatively small aliquot studied, this method should be used
only when rapid "ballpark" numbers are needed. This method, and others
employing similar methodology, are not recommended for precise, quantitative
analysis of samples.
5.2 Subsampling by splitting
This is the best method for quantitative analysis of zooplankton. Two types
of splitters are recommended: the classic Folsom splitter (McEwen et al.
1954) and the newer Burrell et_ al. (1974) device. The Folsom splitter has
received some criticism, due to variations associated with individual han-
dling, but certain simple modifications can alleviate this situation.
16
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Samples should be sorted according to the following guidelines:
(1) Remove excess formaldehyde; place sample in culture dish with 5 mm
square grid; place dish under dissecting microscope.
(2) Remove any animals that are rare. Look at numbers within groups that
appear in the whole sample. If less than 200 are in any group, remove
all individuals within that group. Keyed laboratory counters prove
useful for recording individual counts. Animals should be placed in
labelled vials of 4% formaldehyde.
(3) Place sample in splitter. Split sample, wash and rinse splitter into
sub-splits. Remove and record all individuals of any groups that
contain a "workable number" of organisms (100-200 individuals).
(4) Continue splitting and sorting in this manner until an entire sample
has been counted.
(5) Splitter should be rinsed with fresh water.
Proper use of a splitter is best illustrated by example:
A 1/2 split contains decapod larvae plus numerous cladocerans and copepods.
Decapods are counted and removed until none are left, a total of 150 being
counted. Sample is then split and it appears that cladoceran numbers are
workable. 175 individuals are counted, removed and recorded. Sample is
split again to count copepods, and 156 individuals are counted.
Numbers in total sample:
Decapod larvae = 150 x 2 = 300
Cladocerans = 175 x 4 = 700
Copepods = 156 x 8 = 1248
In situations where certain groups require greater attention or study the
"workable number" may be increased. Conversely, in situations where certain
groups will receive little study, or when monotypic groups are observed, the
"workable number" may be decreased.
Abundance estimates derived from splitting, and information derived from
flowmeters, facilitate the calculation of abundance/water volume. Further-
more, fiducial limits can be placed on numbers within groups that have "been
counted. After samples have been sorted into vials, specialists can deter-
mine the species compositions of individual groups.
Other methods for separating organisms by size groups, and density patterns
have shown promise for sorting certain individual groups.
5.3 Biomass
5.3.1 Introduction
Within the natural environment, every trophic level contains, at any given
time, a fixed amount of living material, composed of several kinds of
organisms. This living material is referred to as the standing crop, the
biomass being a useful expression of it. The most popular techniques for
presenting zooplankton biomass are: (1) Settling volume, (2) Displacement
17
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volume, (3) Wet weight, (4) Dry weight, and (5) Ash-free dry weight. Dis-
placement and settling volume, and wet weight are non-destructive measure-
ments. These methods leave samples intact for further taxonomic and other
uses. They are also less time demanding and require less equipment and skill
than other methods. They do not, however, provide any insight on the nutri-
tional content of the plankton. If this information is desired, dry weight
and ash-free dry weight measurements should be conducted. Ash-free dry weight
is considered the most desirable technique for reporting the chemical (organic
carbon) composition. Conversion factors between biomass techniques are not
always applicable, due to variations in interstitial water content between
different sized samples (Wiebe et al. 1975). Smaller samples appear to retain
a larger percentage of interstitial water than do larger samples.
5.3.2 Shipboard Treatment
In cases where biomass information is being sought, the use of paired zoo-
plankton samplers is recommended. In this way, a sampler required to undergo
biomass analysis is derived from a separate net than is a taxonomic sample.
If only a single net is available, subsampling should be conducted rapidly,
and with a minimum of damage resulting to the plankton. In cases where
organic estimates are required, the sample should be washed and rinsed thor-
oughly in distilled water. Triple glass distilled water is recommended
whenever possible. Samples should be placed in a freezer at -20°C, for
storage. If no shipboard freezer is available, maintenance of samples in
a well insulated container, filled with dry ice, is adequate.
Microzooplankton samples obtained from water bottles or pumping generally do
not provide a great deal of material, therefore wet weight or volume measure-
ments are not recommended. Another problem associated with microzooplankton
is that organisms are generally concentrated together with phytoplankton and
detrital material. Direct counts and size measurements using a microscope
may be a better means for obtaining biomass estimates for microzooplankton.
5.3.3 Settling volume
Settling volume is obtained by allowing plankton to settle in a graduated
cylinder or an Imhoff cone. This method is not recommended, since it exhibits
wide variations in tests that attempt to reproduce results, and does not
compare well with other biomass measures. It is still used by planktologists
because of its simplicity and because it leaves material undamaged. This
method should be used only in situations where rough approximations of
biomass are desired.
5.3.4 Displacement volume
This method measures an equivalent volume of liquid that is displaced by the
sample. Displacement volume may be read by several methods of varying com-
plexity. A simple direct measurement proceeds as follows:
(1) Sample is placed in sieve of mesh size equal to or smaller than net used
in capture.
(2) Sample is allowed to drain, and transferred to a measured volume of
water in a graduated cylinder.
(3) The new volume containing sample + known volume is read.
18
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The displacement volume = new volume minus original measured volume
of water.
Another equally valid method reads the volume of water and the interstitial
fluid after it has been subtracted from the original (known) volume of sample
and fluid.
The more sophisticated Mercury Immersion method provides a better means for
removal of interstitial water but requires considerably more time and skill
than do direct measurements. Nevertheless, this method has received consid-
erable use by plankton researchers, and several modifications of the original
method have been developed (see Beers, 1976). Steps are outlined:
(1) Sample is placed in a chamber with a sintered glass base.
(2) Liquid and interstitial fluid is removed by applying air pressure.
(3) The chamber containing the sample is placed in a dish of mercury,
effectively sealing the underside of the glass base.
(4) The chamber is filled to a certain known level by adding water from a
burette. The volume of water utilized is read.
(5) After the sample has been removed, the chamber is filled with only water.
This volume is read.
Displacement volume = step (5) - step (4), which represents the
difference in the volume of water required to fill the chamber,
with and without plankton. It is usually expressed in ml/m of
water volume sampled.
5.3.5 Wet weight
Wet weight is used to determine the actual weight of the "raw" plankton, once
interstitial water has been removed. However, care must be taken to avoid
loss of body fluids from the organisms. A disadvantage of wet-weight is the
existing degree of variation associated with individual technique. Further-
more in mixed zooplankton samples, individual taxa retain different amounts
of water relative to their organic and inorganic constituency. In deter-
mining wet weight, the following steps should be followed:
(1) Pre-weigh glass jar.
(2) Strain plankton through plankton gauze of a smaller mesh size than used
for capture. Rinse thoroughly with fresh water.
(3) Allow water to drain.
(4) Blot sample on absorbent paper towels until water is no longer absorbed
onto the towels.
(5) Transfer sample to glass jar and weigh.
19
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The wet weight is the difference between the weight of the sample + the
jar, less the weight of the jar (step 5-1). Wet weight is usually
expressed in milligrams (or grams)/m3 of water volume sampled.
5.3.6 Dry weight
Dry weight is a measure of the total plankton weight, after the water content
has been removed. To obtain maximum accuracy, drying should be conducted
without the loss of any volatile organic material. Freeze-drying (lyophili-
zation) is the best method for extracting water and providing an accurate dry
weight value, with minimal biochemical alteration of the sample.
If freeze-drying is not possible, oven-drying at 60°C is recommended. This
temperature provides rapid drying and is still low enough to insure a minimum
of organic loss. The time required for drying may vary, according to the
size of the sample; drying is considered complete when a sample attains a
constant weight (two successive readings are not significantly different).
Dry weight is usually expressed in milligrams (or gms)/m^ of water volume
sampled.
5.3.7 Ash-free dry weight
Ash content (weight), in samples that were freeze-or oven-dried to constant
weight is determined by ashing the sample in a muffle furnace at 500°C.
Weights are taken after sample has been allowed to cool. Ash-free dry weight
is the measure of organic material in the sample, exclusive of all water and
inert organic material. It is calculated as follows:
Ash-free dry weight = dry weight - ash weight. Ash-free weight is
usually expressed in milligrams (or gms)/m3 of water volume sampled.
5.3.8 Large gelatinous forms
Remove from sample and measure separately.
20
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SECTION 6
ADDITIONAL BIOCHEMICAL ANALYSES
6.1 Introduction
In recent years there has been a marked increase in the accumulation of data
on the biochemical constituency of marine zooplankton. Procedures for
obtaining total protein, lipid, carbohydrate, ash and chitin values were
summarized by Raymont et al. (1964), and are outlined below.
Values for the individual components are usually expressed as a percentage of
dry weight, obtained from freeze or oven dried samples. Other more detailed
types of analyses are mentioned and references to guide potential investi-
gators are offered.
6.2 Total protein
The largest single fraction of most oceanic plankton is usually the protein.
The most widely accepted method for determining protein from zooplankton is
the colorimetric biuret method.
6.2.1 Biuret method - Procedure:
(1) Homogenize a known dry weight (0-5 mgm) with 1 ml distilled water and
4 ml biuret reagent in a Potter-Elvehjem homogenizer until all purple
particles are dissolved.
(2) Filter homogenate through glass paper. Repeat this step until a clear
filtrate is obtained.
(3) Transfer to a cuvette and read in a spectrophotometer at 540 mu. The
instrument should be zeroed by a blank consisting of 1 ml distilled
water and 4 ml biuret reagent.
The optical density (OD) of the sample should be read against a standard curve
for bovine albumen. The standard curve should be made up by dissolving a
series of known weights (from 0-10 mg) of the albumen in 1 ml distilled water
and 4 ml biuret reagent.
6.2.2 Lowry (et^jal.) method
This is another colorimetric indicator of total protein, and although more
sensitive than the biuret method, it is generally considered a micro-method.
Its use is recommended in situations where only very small amounts of material
are available.
21
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6.3 Total lipid analysis
The lipid is generally the most variable fraction of the marine zooplankton.
Constituent classes of this fraction are important as energy reserves and are
essential for reproductive and structural components. A common procedure used
for calculating total lipid employs extractive and gravimetric methodology.
(1) Lipid is extracted from about 10 mg of dry weight with a 2:1 chloroform-
methanol (V/V) mixture in a Potter-Elvehjem homogenizer.
(2) After filtering, the filtrate is washed with a 0.05 N potassium chloride
solution. Volume of wash solution added should be approx. 20% of the
homogenate volume .
(3) Allow phases to separate by standing (or centrifugation) .
(4) Siphon off upper phase.
(5) Transfer lower phase to pre-weighed container and evaporate solvent in
a stream of nitrogen.
(6) Only lipid should remain. Record weight and express as a percentage of
the initial dry weight.
Modifications of this method using benzene instead of chloroform, and heating
samples in a boiling water bath, have been reported to yield accurate results
for lipids of individual species. Furthermore, colorimetric procedures also
appear adequate.
6.4 Carbohydrate
Carbohydrate has been shown to appear in relatively small amounts in most
marine zooplankton. Values are. almost always less than 4% of the dry weight.
Colorimetric methodology for determination of carbohydrates is recommended.
Procedure :
(1) Place a constant dry weight (1-5 mg) in a boiling tube, with 1 ml
distilled water, 1 ml 5% phenol solution and 5 ml cone.
(2) After allowing to cool for 20 minutes, read optical density (OD) in a
spectrophotometer at 490 m y.
(3) Compare CD's with standard curve for glucose.
6.5 Ash
Obtained from ash-free dry weight analysis (Sec. 5.3.7). Value is material
that remains in muffle furnace after sample has been ashed at 500°C.
22
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6.6 Chitin
Crustacean exoskeletons are chitinous, making this material an important
fraction to quantify. Procedure is as follows:
(1) A known wet weight (250 mg) is boiled for two hours in 50% NaOH.
(2) Sample is left in solution overnight.
(3) Exoskeletons are removed and washed in dilute HC1.
(4) Exoskeletons are washed in water.
(5) Dry and weigh. This material is ash plus chitin.
(6) Ash in a muffle furnace to burn off any organic material. Only ash
should remain.
The chitin value is the difference between the weight of the exoskeleton plus
the ash, and only the ash (step 5 - step 6), and can be expressed as a per-
centage of dry weight. It must be emphasized that the above described pro-
cedure is effective only with "wet" or "raw" samples. It does not apply to
dried material, since a viscous, gummy solution is produced when the alkaline
solution is added. Bamstedt (1974) suggested a modified methodology for
estimating total chitin that can be used on dried material. The procedure
calls for treatments with IN HC1 in a bath of boiling water, and with 4N
NaOH for 20 minutes, also in a water bath. The material is then washed in
a succession of fluids. Attempts to reproduce Bamstedt's procedure on dried
material in this laboratory (VIMS), proved unsuccessful.
6.7 Other analyses
Amino acid and fatty acid analyses are mentioned briefly but detailed pro-
cedures are not presented. References on respective sections should be
consulted.
6.7.1 Amino acids
Methodology has been developed for quantification of free amino acids and
protein hydrolyzates of certain zooplanktonic crustaceans. Free amino acids
are extracted from lipid-free material with hot distilled water. Proteins
are precipitated with hot trichloroacetic acid and hydrolyzates are prepared
by refluxing with 6N HC1. A column chromatograph and technicon autoanalyzer
yield chromatograms for amino acids and protein hydrolyzates.
6.7.2 Fatty acid analysis
Lipid is extracted from the plankton with chloroform-methanol (see Sec. 5.3).
Thin layer and column chromatography can be used to separate lipid into
principle classes, such as phospholipids, triglycerides, hydrocarbons, wax
esters, etc. Gravimetric analysis has, in certain cases, provided relative
percentages of the various lipid fractions and studies have assessed the
overall significance of these fractions to the organism(s) under examination.
23
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Preparation, hydrogenations and gas liquid chromatography of the methyl
esters of constituent fatty acids has provided insight into the fatty acid
composition of several species of marine and brackish water plankton.
6.8 Biochemistry of marine zooplankton
A bibliography on this rapidly expanding area of investigation is provided,
24
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SECTION 7
DATA ANALYSIS
After samples have been sorted and other appropriate analyses completed, a
cohesive interpretation of data must be developed. Since planktonic organisms
are distributed in patches, classical statistical techniques are of limited
value. Most zooplankton studies to date, have drawn conclusions from obser-
vations of raw data, by ranking abundances of individual species. With
expanding computer usage in recent years, techniques such as diversity
indices and multivariate analyses, have been developed as tools that deter-
mine quantitative relationships among and between zooplankton communities,
and between community and environmental parameters.
7.1 Total numbers and frequency of occurrence
Historically this has been the most popular method of presenting zooplankton
data. Abundances of individual species (or groups) are listed and ranked.
Data on frequency of and periods of occurrence are also calculated. Abundance
data are usually expressed in units of individuals per volume of water (m3,
100 m^, etc.) sampled. Mean total numbers of individual species (or groups)/
up and percentages of individual species' contribution to the entire sample
can also be determined. Confidence limits may be placed on mean numbers of
individuals but, unfortunately, due to avoidance and the patchy nature of the
plankton, these limits generally cover an extremely wide range. Seasonal
trends are usually developed by plotting abundances (no. of ind./m3) VS.
months (or other sampling time period). In addition to baseline and monitor-
ing surveys, studies concerned with day-night differences, spatial variations
and ecological zonation of zooplankton communities have employed this type
of data interpretation. It is still the most popular, and in many cases,
the most meaningful interpretation of data.
7.2 Diversity indices
Diversity indices can be used to establish species diversity gradients in
geographical areas. These indices allow for comparisons between community
diversity in different areas, with respect to parameters selected (i.e.
effects of seasonality, coastal vs. offshore populations of copepods).
Diversity indices can also be used as a baseline statistic, in areas where
natural or man made changes are anticipated, and can be useful in determining
effects of pollutants on changing zooplankton community structure.
These indices are applicable only after the number of species, and the total
25
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number of individuals within a species, have been tabulated. Diversity
indices can then offer a statement on the "information content" of each
individual in a sample. The Shannon-Wiener H', has been a most widely
used diversity statistic and is calculated as follows:
H1 = - Zp
where p. = number of individuals in the ith species
total number of individuals
The index weighs both numbers of species and evenness of species distri-
butions. In systems of low diversity, each successive individual drawn
from a sample would, since it is likely to be the same species as a previously
drawn individual, contribute rather little to the information of the system;
adequate knowledge could be obtained after relatively few drawings. This
would result in a low H' value, since individual information input is low.
In diverse systems, each successive individual is likely to be different
from one previously drawn, and makes a significant contribution to the
knowledge of the system. Many drawings would be required to understand
the system, and the diversity statistic H' would be high.
Criticisms and modifications in the application of diversity indices have been
widespread in the literature. One method claims that the value of H1 can be
improved by scaling all measurements down to a common sampling size. Other
investigators question the value and validity of these indices for ecological
research.
Very often, pollution in present day ecosystems results in an overall reduc-
tion of certain species and the elimination of others; a few species are,
however, capable of exploiting these conditions. Diversity indices may
prove useful in pollution-related studies. However, diversity measurements
as indicated by H' reflect changes in overall information content rather
than enumerate upon an individual species composition changes. In a sit-
uation where one species is completely replaced by another, the diversity
value would remain constant, yet the change of the species (or several spe-
cies) may be indicative of significant environment modifications. In cases
like this, use of an index of faunal overlap may prove helpful. Of the
numerous overlap indices available, a commonly used measurement is the
percentage similarity statistic, which ranges from 0 when two samples
contain no species in common, to 100 when two samples are identical in
both species and abundances.
PS = 100 (1 - 0.5 E|pia - pib|)
where p. = total number of individuals in the ith species in sample
a f total number of individuals in sample a.
p., = the same for sample b.
Thus, diversity indices used in conjunction with either other indices and
statistics, or associated observations can, in certain cases, provide an
26
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effective tool in analyzing aspects of zooplankton community structure.
7.3 Multivariate analysis
This type of analysis draws its conclusions by recognizing patterns between
variables. As data sets increase in size, the individual's ability to detect
trends or patterns naturally declines. The increase in availability and
usage of computers has made these analyses feasible and desirable. These
techniques condense multidimensional relationships by projecting data onto
a reduced number of planes. Thus the multivariate approach tests the reality
of a variable, by expressing its variability in terms of an additional coord-
inate and seeing whether the variable displays a systematic and logical
pattern.
Cluster analysis is a multivariate approach that orders sets of taxa on the
basis of pre-determined criteria. For example, data from zooplankton surveys
may cluster and distinguish groups of samples based upon similarities in
composition. The inverse analysis clusters the species according to simi-
larities in their distribution and abundances. Other multivariate approaches,
referred to as ordination, make no assumptions in grouping the entities or
in drawing boundaries between classes. Recently, principal component anal-
ysis has been used to describe and quantify the major elements responsible
for fluctuations in zooplankton abundances (Colebrook 1977).
27
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SECTION 8
SECTIONAL BIBLIOGRAPHY
2.2 Spatial variations
Anraku, M. 1956. Some experiments on the variability of horizontal plankton
hauls and the horizontal distribution of plankton in a limited area. Bull.
Fac. Fish. Haddaido Univ. 7(1):1-16.
Anraku, M. 1975. Microdistribution of marine copepods in a small inlet.
Mar. Biol. 30:79-87.
Barnes, H. 1949a. On the volume measurement of water filtered by a plankton
pump, with some observations on the distribution of planktonic animals. J.
nar. biol. Ass. U.K. 28:651-661.
Barnes, H. 1949b. A statistical study of the variation in vertical plankton
hauls, with special reference to the loss of the catch with divided hauls.
J. mar. biol. Ass. U.K. 28(2):429-446.
Barnes, H. and S. M. Marshall. 1951. On the variability of replicate
plankton samples and some applications of contagious series to the statistical
distribution of catches over restricted periods. J. mar. biol. Ass. U.K.
30:233-263.
Boyd, C. M. 1973. Small scale spatial patterns of marine zooplankton
examined by an electronic in situ zooplankton detecting device. Neth. J.
Sea Res. 7:103-111.
Cassie, R. M. 1960. Factors influencing the distribution of plankton in the
mixing zone between oceanic and harbour waters. N.Z. J. Sci. 3:26-50.
Cassie, R. M. 1963. Microdistribution of plankton. Oceanog. Mar. Biol.
Ann. Rev. 1:223-252.
Gushing, D. H. 1962. Patchiness. Rappt. Proces-Verbaux Reunions, Conseil
perm. int. Explor. Mer. 15:152-163.
Fasham, J. J. R., M. V. Angel & H. S. J. Roe. 1974. An investigation of the
spatial pattern of zooplankton using the Longhurst-Hardy plankton recorder.
J. exp. mar. Biol. Ecol. 16:93-112.
Haeckel, E. 1890. Plankton studies [transl. by G. W. Field]. Rep. U.S.
Comm. Fish. 1889-1891, p. 565-641.
28
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Harder, W. 1968. Reactions of plankton organisms to water stratification.
Limnol. Oceanogr. 13:156-168.
Hardy, A. C. 1936. Observations on the uneven distribution of the oceanic
plankton. Discovery Rep. 11:511-538.
Hardy, A. C. 1956. The open sea, Pt. 1. Houghton Mifflin. 335 p.
Haury, L. R. 1976. Small-scale patterns of a California Current zooplankton
assemblage. Mar. Biol. 37:137-157.
Longhurst, A. R. 1967. Diversity and trophic structure of zooplankton com-
munities in the California Current. Deep-Sea Res. 14:393-407.
Smith, L. R., C. B. Miller & R. L. Holton. 1976. Small-scale horizontal
distribution of coastal copepods. J. exp. mar. Biol. Ecol. 23:241-253.
Wiebe, P. H. 1970. Small-scale spatial distribution in oceanic zooplankton.
Limnol. Oceanogr. 15:205-217.
Wiebe, P. H. 1971. A computer model of zooplankton patchiness and its
effects on sampling error. Limnol. Oceanogr. 16:29-38.
Wiebe, P. H. and W. R. Holland. 1968. Plankton patchiness: effects on
repeated net tows. Limnol. Oceanogr. 13:315-320.
2.3 Temporal variations
Backus, R. H., J. E. Craddock, R. L. Haedrich and D. L. Shores. 1970. The
distribution of mesopelagic fishes in the equatorial and western North
Atlantic Ocean. J. Mar. Res. 28(2):179-201.
Badcock, J. 1970. The vertical distribution of mesopelagic fishes collected
on the Sond cruise. J. mar. biol. Ass. U.K. 50:1001-1044.
Barham, E. G. 1970. Deep-sea fishes: lethargy and vertical orientation.
In Biological sound scattering in the ocean, pp. 100-118. Ed. G. B. Farquhar.
Maury Center for Ocean Science Rep. 005. U.S. Government Printing Office.
Brinton, E. 1962. Variable factors affecting the apparent range and esti-
mated concentration of euphausiids in the North Pacific. Pacif. Sci. 16(4):
374-408.
Clarke, T. A., and P. J. Wagner. 1976. Vertical distribution and other
aspects of the ecology of certain mesopelagic fishes -taken near Hawaii.
Fish. Bull. 74(3):635-645.
Deevey, G. B. 1971. The annual cycle in quantity and composition of the
zooplankton of the Sargasso Sea off Bermuda. I. The upper 500 M. Limnol.
Oceanogr. 18:219-240.
29
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Fager, E. W. and J. A. McGowan. 1963. Zooplankton species groups in the
North Pacific. Science 40:453-460.
Hardy, A. C. 1935. The plankton community, the whole fisheries and the
hypothesis of animal exclusion. Discovery Rep. 11:273-370.
Haury, L. R. 1976. Small-scale patterns of a California Current zooplankton
assemblage. Mar. Biol. 37:137-157.
Jacobs, F. & G. C. Grant. MS. Seasonal composition and biomass of zooplank-
ton in the lower Chesapeake Bay. In prep.
Maynard, S. D., F. V. Riggs and J. F. Walters. 1975. Mesopelagic micronekton
in Hawaiian waters: faunal composition, standing stock, and diel migration.
Fish. Bull. 73:726-736.
Miller, C. B. 1970. Some environmental consequences of vertical migration
in marine zooplankton. Limnol. Oceanogr. 15:727-741.
Pearcy, W. G. 1964. Some distributional features of mesopelagic fishes off
Oregon. J. Mar. Res. 22:83-102.
Platt, T. and R. J. Conover. 1971. Variability and its effect on 24 h
chlorophyll budget of a small marine basin. Mar. Biol. 10:52-65.
Raymont, J. E. G. 1963. Plankton and productivity in the oceans. Pergamon
Press. 660 p.
Russell, F. S. 1927. The vertical distribution of plankton in the sea.
Biol. Rev. 2:213-256.
Russell, F. S. 1928. The vertical distribution of marine macroplankton.
VII. Observations on the behaviour of Calanus finmarchicus. J. mar. biol.
Ass. U.K. 15:429-454.
Russell, F. S. 1931. The vertical distribution of marine macroplankton. X.
Notes on the behaviour of Sagitta in the Plymouth area. J. mar. biol. Ass.
U.K. 17:391-414.
Russell, F. S. 1934. The vertical distribution of marine macroplankton.
XII. Some observations on the vertical distribution of Calanus finmarchicus
in relation to light intensity. J. mar. biol. Ass. U.K. 19:569-584.
Sameoto, D. D. 1975. Tidal and diurnal effects on zooplankton sample vari-
ability in a nearshore marine environment. J. Fish. Res. Bd. Can. 32:347-366.
Youngbluth, M. J. 1975. The vertical distribution and diel migration of
euphausiids in the central waters of the eastern South Pacific. Deep-Sea
Res. 22:519-536.
30
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2.4 Introduction to bias and variation associated with sampling
Biological Methods Panel Committee on Oceanography. 1969. Recommended pro-
cedures for measuring the productivity of plankton standing stock and related
oceanic properties. National Acad. of Sciences, Washington, D. C. 59 pp.
Clutter, R. I. and M. Anraku. 1968. Avoidance of samplers. pp. 57-76. In;
D. J. Tranter [Ed.] Zooplankton sampling. UNESCO. Monographs on oceano-
graphic methodology. No. 2. 174 p.
Laval, P. H. 1974. Un modele mathematique de 1'evitement d'un filet a
plancton, son application pratique, et sa verification indirected en
recourant an parasitisme de 1'amphipode hyperide Vebilia armata Bouvallius.
J. exp. mar. Biol. Ecol. 14:57-87.
Singarajah, K. V. 1969. Escape reactions of zooplankton: the avoidance of
a pursuing siphon tube. J. exp. mar. Biol. Ecol. 3:171-178.
3.1 Sampling location and station selection
Biological Methods Panel Committee on Oceanography (E. Ahlstrom, Chairman).
1969. Recommended procedures for measuring the productivity of plankton
standing stock and related oceanic properties. National Acad. of Sciences,
Washington, D. C. 59 pp.
Dicks, B. 1976. Offshore biological monitoring. Pp. 325-359. In J. M.
Baker (Ed.). Marine ecology and oil pollution. Proceedings of a meeting in
Aviemore, Scotland, Apr. 1975, Halstead (Wiley) N. Y. 566 pp.
Volkman, G., J. Krauss and A. Vine. 1956. The use of parachute drogue in
the measurement of subsurface ocean currents. Trans. Am. Geophys. Union
37:573-577.
3.2.1 Water bottles
Biological Methods Panel Committee on Oceanography (E. Ahlstrom, Chairman).
1969. Recommended procedures for measuring the productivity of plankton
standing stock and related oceanic properties. National Acad. of Sciences,
Washington, D. C. 59 pp.
UNESCO. 1968. Microzooplankton. Report of working party No. 1. pp. 150-
152. In: D. J. Tranter (Ed.) Zooplankton sampling. Monographs on oceano-
graphic methodology, No. 2. 174 p.
3.2.2 Pumping systems
Beers, J. R. and G. L. Stewart. 1967. Micro-zooplankton in the euphotic
zone at five locations across the California Current. J. Fish. Res. Bd.
Can. 24:2053-2068.
31
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Beers, J. R. and G. L. Stewart. 1969. The vertical distribution of micro-
zooplankton and some ecological observations. J. Cons. perm. int. Explor.
Mer. 33:30-44.
Beers, J. R., G. L. Stewart and J. D. H. Strickland. 1967. A pumping system
for sampling small plankton. J. Fish. Res. Bd. Can. 24:1811-1818.
Cassie, R. M. 1958. Apparatus for investigating spatial distribution of
plankton. N.Z. J. Sci., 1:436-448.
Leong, R. 1969. Evaluation of a pump and reeled hose system for studying
the vertical distribution of small plankton. Spec. Scient. Rep. U.S. Fish.
Wild. Serv. Fish, No. 545, 9 p.
O'Connell, C. P. and R. J. H. Leong. 1963. A towed pump and shipboard
filtering system for sampling small zooplankters. Spec. Scient. Rep. U.S.
Fish. Wild. Serv., Fish., No. 452, 19 pp.
3.2.3 Introduction to net sampling
Biological Methods Panel Committee on Oceanography. 1969. Recommended
procedures for measuring the productivity of plankton standing stock and
related oceanic properties. National Acad. of Sciences, Washington, D.C.
59 pp.
Smith, P. E., R. C. Counts and R. I. Clutter. 1968. Changes in filtering
efficiency of plankton nets under tow due to clogging. J. Cons. perm. int.
Explor. Mer. 32:1-13.
Vannucci, M. 1968. Loss of organisms through the meshes, pp. 77-86. In
D. J. Tranter (Ed.). UNESCO Monographs on Oceanographic Methodology. No. 2.
174 p.
3.2.4 General considerations of net sampling
Aron, W. and S. Collard. 1969. A study on the influence of net speed on
catch. Limnol. Oceanogr. 14:242-249.
Barkley, R. A. 1964. The theoretical effectiveness of towed-net samplers as
related to sampler size and to swimming speed of organisms. J. Cons. perm.
int. Explor. Mer. 29:146-157.
Clutter, R. I. and M. Anraku. 1968. Avoidance of samplers, pp. 57-76.
In: D. J. Tranter (Ed.) Zooplankton sampling. UNESCO Monographs on
oceanographic methodology. No. 2. 174 p.
Fleminger, A. and R. I. Clutter. 1965. Avoidance of towed nets by zooplank-
ton. Limnol. Oceanogr. 11:456-469.
32
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Friedl, W. A. 1971. The relative sampling performance of 6- and 10-foot
Isaacs-Kidd midwater trawls. Fish. Bull. 69:427-432.
Gehringer, J. W. and W. Aron. 1968. Field techniques, pp. 87-104. In
D. J. Tranter [Ed.] UNESCO Monographs on oceanographic methodology. No. 2.
174 p.
Laval, P. H. 1974. Un modele mathematique de 1'evitement d'in filet a
plancton, son application pratique, et sa verification indirecte en
recourrant an parasitime de 1'amphipode hyperide Vibilia armata Bouvallius.
J. exp. mar. Biol. Ecol. 14:57-87.
Le Fevre, J. 1973. Clogging and filtration coefficient in a light-speed
plankton sampler. Mar. Biol. 21:29-33.
McGowan, J. A. and V. J. Fraundorf. 1966. The relationship between size of
net used and estimates of zooplankton diversity. Limnol. Oceanogr. 11:456-
469.
Noble, R. L. 1970. Evaluation of the Miller high-speed sampler for sampling
yellow perch and walleye fry. J. Fish. Res. Bd. Can. 27:1033-1044.
Sameoto, D. D. 1975. Tidal and diurnal effects on zooplankton sample vari-
ability in a nearshore marine environment. J. Fish. Res. Bd. Can. 32:347-366.
Smith, P. E., R. C. Counts and R. I. Clutter. 1968. Changes in filtering
efficiency of plankton nets due to clogging under tow. J. Cons. perm. int.
Explor. Mer. 32:232-248.
Tranter, D. J. and P. E. Smith. 1968. Filtration performance, pp. 27-56.
In D. J. Tranter (Ed.) UNESCO Monographs on Oceanic Methodology. No. 2.
174 p.
Vannucci, M. 1968. Loss of organisms through the meshes, pp. 77-86. In
D. J. Tranter (Ed.) UNESCO Monographs on Oceanographic Methodology. No. 2.
174 p.
Wiebe, P. H. 1972. A field investigation of the relationship between length
of tow, size of net and sampling error. J. Cons. perm. int. Explor. Mer.
34(2):268-275.
Wiebe, P. H. and W. R. Holland. 1968. Plankton patchiness: effects on
repeated net tows. Limnol. Oceanogr. 13:315-321.
3.2.5 Recommended nets for ocean sampling
Baker, A de C., M. R. Clarke and M. J. Harris. 1973. The N.I.O. combination
net (RMT 1+8) and further developments of rectangular midwater trawls. J.
mar. biol. Ass. U.K. 53:167-184.
33
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Brown, N. L. 1974. A precision CTD microprofiler. Proceedings of the
International Conference on Engineering in the Ocean Environment. Halifax,
Nova Scotia, Vol. 2, 270-278.
Bruce, R. H. and J. Aiken. 1975. The undulating oceanographic recorder - a
new instrument system for sampling plankton and recording physical variable
in the euphotic zone from a ship underway. Mar. Biol. 32:85-97.
Fasham, M. J. R., M. V. Angel and H. S. J. Roe. 1974. An investigation of
the spatial pattern of zooplankton using the Longhurst-Hardy Plankton Re-
corder. J. exp. mar. Biol. Ecol. 16:93-112.
Frost, B. W. and L. E. McCrone. 1974. Vertical distribution of zooplankton
and myctophid fish at Canadian weather station P, with description of a new
multiple net trawl. Proceedings of the International Conf. on Engineering
in the Ocean Environment. Halifax, Nova Scotia, vol. 1, 159-165.
Haury, L. R. 1973. Sampling bias of a Longhurst-Hardy Plankton Recorder.
Limnol. Oceanogr. 18:500-506.
Haury, L. R., P. H. Wiebe and S. H. Boyd. 1976. Longhurst-Hardy Plankton
Recorder: their design and use to minimize bias. Deep-Sea Res. 23:1217-1229.
Jossi, J. W. 1970. Annotated bibliography of zooplankton sampling devices.
U.S. Dept. Interior, U.S. Fish and Wildlife Serv. No. 609.
Longhurst, A. R., A. D. Reith, R. E. Bower and L. D. R. Siebert. 1966. A
new system for the collection of multiple serial plankton samples. Deep-Sea
Res. 13:213-222.
McGowan, J. A. and D. M. Brown. 1966. A new opening-closing paired zoo-
plankton net. Univ. Calif. Scripps Inst. Oceanogr. (Ref. 66-23).
Tucker, G. H. 1951. Relation of fishes and other organisms to the scattering
of underwater sound. J. Mar. Res. 10:215-238.
Wiebe, P. H., K. H. Burt, S. H. Boyd and A. W. Morton. 1976. A multiple
opening/closing net and environmental sensing system for sampling zooplankton.
J. Mar. Res. 34(3):313-326.
3.2.6 Care of nets
UNESCO. 1968. Smaller mesozooplankton. Report of working party No. 2.
pp. 153-159. In; D. J. Tranter (Ed.) Zooplankton sampling. Monographs on
oceanographic methodology. No. 2. 174 p.
4. Shipboard handling of samples
34
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Griffiths, F. B., A. Fleminger, B. Kimor and M. Vannucci. 1976. Shipboard
and curating techniques, pp. 17-33. ^n H. F. Steedman [Ed.] UNESCO.
Monographs on oceanographic methodology. No. 4. 350 p.
UNESCO. 1968. Smaller mesozooplankton. Report of working party No. 2, pp.
153-159. In: D. J. Tranter (Ed.) Monographs on oceanographic methodology.
No. 2. 174 p.
4.4 Special cases
Adams, H. R., A. P. Flerchinger and H. F. Steedman. 1976. Ctenophora
fixation and preservation, pp. 270-271. In: H. F. Steedman (Ed.) UNESCO.
Monographs on oceanographic methodology. No. 4. 350 p.
Grice, G. D., G. R. Harvey, V. T. Bowen and R. H. Backus. 1972. The col-
lection and preservation of open ocean marine organisms for pollutant
analysis. Bull. Environmental Contamination and Toxicology 7:125-132.
Hamner, W. M., L. P. Madin, A. L. Alldredge, R. W. Gilmer, and P. P. Hamner.
1975. Underwater observations of gelatinous zooplankton: Sampling problems,
feeding biology and behavior. Limnol. Oceanogr. 20:907-917.
UNESCO. 1976. Part VIII. Fixation and preservation of various marine taxa.
In H. F. Steedman (Ed.), Monographs in oceanographic methodology, No. 4.
350 p.
5.1 Subsampling by pipette method
Frolander, H. F. 1968. Statistical variation in zooplankton numbers from
subsampling with a Stempel pipette. J. Wat. Pollut. Control Fed. 40, R82-
R88.
Weber, C. 1973. Biological field and laboratory method for measuring the
quality of surface waters and effluents. Nat. Environmental Res. Center
Office of Res. Development. U.S. Environmental Protection Agency. 20 p.
5.2 Subsampling by splitting
Be', A. W. H. 1959. A method for rapid sorting of Foraminifera from marine
plankton samples. J. Paleontol. 33:846-848.
Bowen, R. A., J. M. St. Onge, J. B. Colton and C. A. Price. 1972. Density-
gradient centrifugation as an aid to sorting planktonic organisms. I.
Gradient materials. Mar. Biol. 14:242-247.
Burrell, V. G., W. A. Van Engel, and S. G. Hummel. 1974. A new device for
subsampling plankton samples. J. Cons. perm. int. Explor. Mer. 35:364-366.
35
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Furnass, T. I. and W. C. Findley. 1975. An improved sorting device for
zooplankton. Limnol. Oceanogr. 20:295-297.
Horwood, J. W. and R. M. Driver. 1976. A note on a theoretical subsampling
distribution of macroplankton. J. Cons. perm. int. Explor. Mer. 36(3):274-
276.
Kirkerud, L. A. 1974. A method for the rapid sorting of plankton into a
number of size groups. J. Cons. perm. int. Explor. Mer. 35:367-369.
Longhurst, A. R. and D. L. R. Siebert. 1967. Skill in the use of Folsom's
plankton sample splitter. Limnol. Oceanogr. 12:334-335.
McEwen, G. F., M. W. Johnson and T. R. Folsom. 1954. A statistical analysis
of the performance of the Folsom plankton sample splitter based upon test
observations. Arch. Meteorol. Geophys. Bioklimatol., Ser. A Meleorol.
Geophys. 7:502-527.
McGowan, J. A. and V. J. Fraundorf. 1964. A modified heavy fraction zoo-
plankton sorter. Limnol. Oceanogr. 9:152-154.
Nival, S. and P. Nival. 1971. A simple device for the sorting of living
planktonic copepods. Limnol. Oceanogr. 16:977-980.
Venrick, E. L. 1971. The statistics of subsampling. Limnol. Oceanogr. 16:
811-818.
5.3 Biomass
Ahlstrom, E. H. and J. R. Thrailkill. 1963. Plankton volume loss with time
of preservation. Calif. Coop. Oceanic Fish. Invest. Rep. 9:57-73.
Beers, J. R. 1976. Determination of zooplankton biomass. Pp. 35-84. In
H. F. Steedman [Ed.] Zooplankton fixation and preservation. UNESCO. Mon-
ographs on oceanographic methodology. No. 4. 350 p.
Curl, H., Jr. 1962. Analyses of carbon in marine plankton organisms. J.
Mar. Res. 20:181-188.
Fudge, H. 1968. Biochemical analysis of preserved zooplankton. Nature
219:380-381.
Harris, R. H. 1976. Freeze drying, pp. 97-99. In: H. F. Steedman [Ed.],
Zooplankton fixation and preservation. UNESCO. Monographs on oceanographic
methodology. No. 4. 350 p.
Howey, T. W. 1976. Zooplankton of the Gulf of Mexico. Distribution of
displacement volume, occurrence of systematic groups, abundance and diversity
among copepods. Dissertation L.S.U. 93 p.
36
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Lovegrove, T. 1962. The effect of various factors on dry weight values.
Rapp. Cons. Explor. Her. 153:86-91.
Lovegrove, T. 1966. The determination of the dry weight of plankton and
the effects of various factors on the values obtained, pp. 429-467. In:
H. Barnes (Ed.). Some contemporary studies in marine science. London,
Allen and Univinn Ltd. 716 p.
Nakai, Z. 1955. The chemical composition, volume, weight and size of the
important marine plankton. Spec. Publs Tokai Fish. Res. Lab. 5:12-24.
Nakai, Z. and K. Honjo. 1962. Comparative studies on measurements of the
weight and the volume of plankton samples. A preliminary account. Indo-Pac.
Fish Counc. Proc., 9th Sess., Sec. II, p. 9-16.
Platt, T., M. Brown and B. Irvin. 1969. Caloric and carbon equivalents of
zooplankton biomass. J. Fish. Res. Bd. Can. 26:2345-2349.
Platt, T. and B. Irvin. 1973. Caloric content of phytoplankton. Limnol.
Oceanogr. 18:306-310.
Smith, R. L. 1966. Ecology and field biology. Harper and Rowe. New York.
686 p.
Steedman, H. F. 1974. Laboratory methods in the study of marine zooplankton.
J. Cons. perm. int. Explor. Mer., 35:351-358.
Utermohl, H. 1958. Zur Vervollkommuny der quantitativen Phytoplankton.
Methodik. Mitt. d. Int. Vereinig f. Limnologie, no. 9, 39 p.
van Heusden, G. P. H. 1972. Estimation of the biomass of plankton.
Hydrobiologia 39:165-208.
Wiebe, P. H., K. H. Burt, S. H. Boyd and J. L. Cox. 1975. Relationships
between zooplankton displacement volume, wet weight, dry weight, and carbon.
Fish. Bull. 73(4):777-786.
Yentsch, C. S., and J. E. Hebard. 1957. A gauge for determining plankton
volume by the mercury immersion method. J. Cons. perm. int. Explor. Mer.,
22:184-190.
6.2 Total protein analysis
Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.
Protein measurement by the Folin-phenol reagent. J._ Biol. Chem. 196:
265-275.
Parvin, R., S. V. Parde and T. A. Venkitasubramanian. 1965. On the color-
imetric biuret method of protein determination. Analyt. Biochem. 12:219-229.
37
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Raymont, J. E. G., J. Austin and E. Linford. 1964. Biochemical studies on
marine zooplankton. I. The biochemical composition of Neomysis integer.
J. Cons. perm. int. Explor. Her. 28:354-363.
6.3 Total lipid analysis
Armenta, J. S. 1964. A rapid chemical method for quantification of lipids
separated by thin-layer chromatography. J. Lipid Res. 5:270-272.
Bligh, E. G. and W. J. Dyer. 1959. A rapid method of total lipid extraction
and purification. Chr. J. Biochem. Physiol. 37:911-917.
Donaldson, H. A. 1976. Chemical composition of sergestid shrimps (Decapoda:
Natantia) collected near Bermuda. Mar. Biol. 38:51-58.
Fisher, L. R. 1962. The total lipid material in some species of marine
zooplankton. Rapp. Cons. Explor. Her. 153:129-136.
Folch, J., M. Lees and G. H. Sloane Stanley. 1957. A simple method for the
isolation and purification of total lipids from animal tissues. J. biol.
Chem. 226:646-648.
Metcalfe, L. D. and A. A. Schmitz. 1961. The rapid preparation of fatty
acid esters for gas chromatographic analysis. Anal. Chem. 33:363-364.
Raymont, J. E. G., J. Austin and E. Linford. 1964. Biochemical studies on
marine zooplankton. I. The biochemical composition of Neomysis integer.
J. Cons. perm. int. Explor. Mer. 28:354-363.
Zb'llner, N. and K. Kirsch. 1962. Uber die quantitative Bestimmuny von
Lipoider (Mikromethode) mittels den vielen naturllchen Lipoiden (Allen
bekannten Plasmalipoiden) gemeinsamen Sulfophosphovanillen - Reaktion.
Zectschrift fur die gesamte exper. Med. 135:545-561.
6.4 Carbohydrate analysis
Dubois, M., K. A. Gills, J. K. Hamilton, P. A. Reebers, and F. Smith. 1956.
Colorimetric method for determination of sugars and related substances.
Anal. Chem. 28:350-356.
Krey, J. 1950. Eine neue Methode Zur quantitativen Bestimmuny des Plankton.
Kieler Meeresforsch 7:58-75.
Mendel, B., A. Kemp and D. K. Myers. 1954. A colorimetric micromethod for
the determination of glucose. J. Biochem. 56:639-46.
Raymont, J. E. G., J. Austin and E. Linford. 1964. Biochemical studies on
marine zooplankton. I. The biochemical composition of Neomysis integer.
J. Cons. perm. int. Explor. Mer. 28:354-363.
38
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6.5 Ash analysis (See also Sec. 5.3.7)
Raymont, J. E. G., J. Austin and E. Linford. 1964. Biochemical studies on
marine zooplankton. I. The biochemical composition of Neomysis integer.
J. Cons. perm. int. Explor. Her. 28:354-363.
6.6 Chitin analysis
Baumstedt, V. 1974. Biochemical studies on the deep-water pelagic community
of Korsfjorden, Western Norway. Methodology and sample design. Sarsia 56:
71-86.
Raymont, J. E. G., J. Austin and E. Linford. 1964. Biochemical studies on
marine zooplankton. I. The biochemical composition of Neomysis integer.
J. Cons. perm. int. Explor. Her. 28:354-363.
6.7 Other analyses
American Oil Chemists' Society. 1965. Official and tentative methods.
Chicago, 111.
Culkin, F. and R. J. Morris. 1969. The fatty acids of some marine crus-
taceans. Deep. Sea. Res. 16:109-116.
Culkin, F. and R. J. Morris. 1970. The fatty acids of some cephalopods.
Deep-Sea Res. 17:171-174.
Folch, J., M. Lees and G. H. Sloane Stanley. 1957. A simple method for the
isolation and purification of total lipids from animal tissues. J. biol.
Chem. 226:497-509.
Klein, F. K. and H. Rapoport. 1970. A two-dimensional pipette for sample
application in preparative thin-layer chromatography. J. Chromatography
47:505-506.
Morrison, W. R. and L. M. Smith. 1964. Preparation of fatty acid methyl
esters and dimethylacetates from lipids with boron fluoride-methanol. J.
Lipid Res. 5:600-608.
Raymont, J. E. G., J. Austin and E. Linford. 1968. Biochemical studies on
marine zooplankton. V. The composition of the major biochemical fractions
in Neomysis integer. J. mar. biol. Ass. U.K. 48:735-760.
Srinivasagam, R. T., J. E. G. Raymont, C. F. Woodie,_and J. K. B. Raymont.
1971. Biochemical studies on marine zooplankton. X. The amino acid
composition of Euphausia superba, Meganyctiphanes norvegica and Neomysis
integer. J. mar. biol. Ass. U.K. 51:917-925.
39
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6.8 The biochemistry of zooplankton
Ackman, R. G. and C. A. Eaton. 1966. Lipids of the fin whale (Balaenoptera
physalus) from North Atlantic waters. III. Occurrence of eicosenoic and
docosenoic fatty acids in the zooplankter Meganyctiphanes norveigca (M. Sears)
and their effect on whale oil composition. Can. J. Biochem. 44:1561-1566.
Ackamn, R. G. and C. A. Eaton. 1967. Fatty acid composition of the decapod
shrimp Pandalus borealis in relation to that of the euphausid Meganyctiphanes
norvegica. J. Fish. Res. Bd. Can. 24:467-471.
Ackman, R. G., C. S. Tocher and J. McLachlan. 1968. Marine phytoplankter
fatty acids. J. Fish. Res. Bd. Can. 25:1603-1620.
Ackman, R. G., C. A. Eaton, J. C. Sipos, S. N. Hooper and J. D. Castell.
1970. Lipids and fatty acids of two species of North Atlantic krill
(Meganyctiphanes norvegica and Thysanoessa inermis) and their role in the
aquatic food web. J. Fish. Res. Bd. Can. 27:513-533.
Armenta, J. S. 1964. A rapid chemical method for quantification of lipids
separate by thin-layer chromatography. J. Lipid Res. 5:270-272.
Barnes, H. , M. Barnes, and D. M. Finlayson. 1963. The seasonal changes in
body weight, biochemical composition, and oxygen uptake of two common boreo-
arctic cirripedes, Balanus balanoides and B. balanus. J. mar. biol. Ass.
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Holm-Hansen, 0. 1968. Determination of particulate organic nitrogen.
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Jeffries, H. P. 1970. Seasonal composition of temperate plankton commun-
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Johannes, R. E., S. J. Coward and K. L. Webb. 1969. Are dissolved amino
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Johannes, R. E. and K. L. Webb. 1965. Release of dissolved amino acids by
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Johannes, R. E. and K. L. Webb. 1970. Release of dissolved organic compounds
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Klem, A. 1932. Contributions to the study of the oils of marine Crustacea.
I. The oils of Meganyctiphanes norvegica, M. Sara and Calanus finmarchicus
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Krey, J. 1950. Eine neue Methode Zur quantitativen Bestimmung des
Planktons. Kieler Meeresforsch 7:58-75.
Krishnaswamy, S., J. Tundisi and J. E. G. Raymont. 1966. The measurement
of succinic dehydrogenase activity in ascidians. Naturwiss. 17, 441, 1-2.
Krishnaswamy, S., J. Tundisi and J. E. G. Raymont. 1967. Succinic
dehydrogenase activity in marine animals. Int. Revue ges. Hydrobiol. 52:
447-451.
Kryuchkova, M. I., and 0. E. Makarov. 1969. Technochemical characteristics
of krill. Trudy vues nachno-issled. Inst. morsk. ryb. Khoz. Okeanogr.
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Lee, R. F., and J. Hirota. 1973. Wax esters in tropical zooplankton and
nekton and the geographical distribution of wax esters in marine copepods.
Limnol. Oceanogr. 18:227-239.
Lee, R. F., J. Hirota and A. M. Barnett. 1971. Distribution and importance
of wax esters in marine copepods and other zooplankton. Deep-Sea Res. 18:
1147-1165.
Lee, R. F., J. C. Kevenzel and G. A. Paffenhdfer. 1970. Wax esters in marine
copepods. Science 167:1510-1511.
Lee, R. F., J. C. Nevenzel, and G. A. Paffenhofer. 1971. Importance of wax
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Lee, R. F. J. Nevenzel, G. A. Paffenhofer and A. A. Benson. 1970. The
metabolism of wax esters and other lipids by the marine copepod, Calanus
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Lewis, R. W. 1967. Fatty acid composition of some marine animals from
various depths. J. Fish. Res. Bd. Can. 24:1101-1115.
Lewis, R. W. 1969. The fatty acid composition of Arctic marine phyto-
plankton and zooplankton with special reference to minor acids. Limnol.
Oceanogr. 14:35-40.
Linford, E. 1965. Biochemical studies on marine zooplankton. II. Vari-
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Littlepage, J. L. 1964. Seasonal variation in lipid content of two
Antarctic marine crustaces. Biol. Antarctique, Actual. Scient. Ind.
1312:463-470.
Lockwood, A. P. M. 1968. Aspects of the Physiology of Crustacea. Oliver
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Lovegrove, T. 1962. The effect of various factors on dry weight values.
Rapp. Cons. Explor. Mer 153:86-91.
Lovegrove, T. 1966. The determination of the dry weight of plankton and
the effect of various factors on the values obtained. In: Some Contemporary
Studies in Marine Science (ed. H. Barnes), George Allen and Unwin, London,
716 pp.
Lovern, J. A. 1935. Fat metabolism in fishes. VI. _The fats of some
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Lovern, J. A. 1964. The lipids of marine organisms. Ann. Rev. Oceanogr.
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Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein
measurement by the Folin-Phenol Reagent. J. Biol. Chem. 196:265-275.
Manwell, C., C. M. Baker, P. Ashton and E. 0. Corner. 1967. Biochemical
differences between Calanus finmarchicus and _C. helgolandicus. Esterases,
malate, and triosephosphate, dehydrogenase, aldolase, 'peptidases' and other
enzymes. J. mar. biol. Ass. U.K. 47:145-169.
Marshall, S. M. and A. P. Orr. 1955. On the biology of Calanus finmarchicus.
J. mar. biol. Ass. U.K. 34:495-529.
Marshall, S. M. and A. P. Orr. 1962. Feeding and digestion in marine
copepods. UNESCO Conference on Radiosotopes in Scientific Research.
UNESCO/NS/RIC/137 6 pp.
Mayzaud, P. 1973. Respiration and nitrogen excretion of zooplankton. II.
Studies of the metabolic characteristics of starved animals. Mar. Biol. 21:
19-28.
Mendel, B., A. Kemp and D. K. Myers. 1954. A colorimetric micro-method for
the determination of glucose. J. Biochem. 56:639-646.
Metcalfe, L. D. and A. A. Schmitz. 1961. The rapid preparation of fatty
acid esters for gas chromatographic analysis. Anal. Chem. 33:363-364.
Morris, R. J. 1971. Seasonal and environmental effects on the lipid compo-
sition of Neomysis integer. J. mar. biol. Ass. U.K. 51(1):21-32.
Morris, R. J. 1973a. Relationships between the sex and degree of maturity
of marine crustaceans and their lipid compositions. J. mar. biol. Ass. U.K.
53:27-37.
Morris, R. J. 1973b. Changes in the lipid composition of Acantephyra
purpurea Milne Edwards (Crustacea:Decapoda) during its diurnal migration:
a preliminary investigation. J. exp. mar. Biol. Ecol. 13:550-61.
Morris, R. J., C. F. Ferguson and J. E. G. Raymont. 1973. Preliminary
studies on the lipid metabolism of Neomysis integer, involving labelled
feeding experiments. J. mar. biol. Ass. U.K. 53:657-664.
Morris, R. J. and J. R. Sargent. 1973. Studies on the lipid metabolism of
some oceanic crustaceans. Mar. Biol. 22:77-83.
Nakai, Z. 1955. The chemical composition, volume, weight and size of the
important marine plankton. Spec. PubIs. Tokai Fish. Res. Lab. 5:12-24.
Nevenzel, J. C. 1970. Occurrence, function and biosynthesis of wax esters
in marine organisms. Lipids 5:308-319.
Omori, M. 1969. Weight and chemical composition of some important oceanic
zooplankton in the North Pacific Ocean. Mar. Biol. 3:4-10.
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Orr, A. P. 1934a. The weight and chemical composition of Euchaeta norvegica
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Orr, A. P. 1934b. On the biology of Calanus finmarchicus. Part IV. Sea-
sonal changes in the weight and chemical composition in Loch Fyne. J. mar.
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Parvin, R. , S. V. Fade and T. A. Venkitasubramanian. 1965. On the color-
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Petipa, T. S. 1966. Oxygen consumption and food requirement in copepods
Acartia clausi Giesbricht and A_. lalisetosa Kritoz. Zool. Zhurnal 45:363-70.
Petipa, T. S., E. V. Pavlova and G. N. Mironov. 1970. The food web struc-
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communities. In: Marine Food Chains (ed. J. H. Steele), pp. 142-167.
Raymont, J. E. G., J. Austin and E. Linford. 1964. Biochemical studies on
marine zooplankton. I. The biochemical composition of Neomysis integer.
J. Cons. perm. int. Explor. Her 28:354-363.
Raymont, J. E. G., J. Austin and E. Linford. 1966. Biochemical studies on
marine zooplankton. III. Seasonal variation in the biochemical composition
of Neomysis integer. In Some Contemporary Studies in Marine Science, ed. H.
Barnes, pp. 597-605. London.
Raymont, J. E. G., J. Austin and E. Linford. 1967. Biochemical composition
of certain oceanic zooplanktonic decapods. Deep-Sea Res. 14:113-115.
Raymont, J. E. G., J. Austin and E. Linford. 1968. Biochemical studies on
marine zooplankton. V. The composition of the major biochemical fractions
in Neomysis integer. J. mar. biol. Ass. U.K. 48:735-760.
Raymont, J. E. G. and R. J. Conover. 1961. Further investigations on the
carbohydrate content of marine zooplankton. Limnol. Oceanogr. 6:154-164.
Raymont, J. E. G. and S. Krishnaswamy. 1960. Carbohydrate in some marine
planktonic animals. J. mar. biol. Ass. U.K. 39:239-248.
Raymont, J. E. G., S. Krishnaswamy and J. Tundisi. 1967. Biochemical
studies on marine zooplankton. IV. Investigations on succinic dehydrogenase
activity in zooplankton with special reference to Neomysis integer. J. cons.
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Raymont, J. E. G. and E. Linford. 1966. A note on the biochemical compo-
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485-488.
Raymont, J. E. G., R. J. Morris, C. F. Ferguson and J. K. D. Raymont. 1975.
Variation in the amino-acid composition of lipid-free residues of marine
animals from the northeast Atlantic. J. exp. mar. Biol. Ecol. 17:261-267.
47
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Raymont, J. E. G., R. T. Srinlvasagam, and J. K. B. Raymont. 1969a.
Biochemical studies on marine zooplankton. VI. Investigations on
Meganyctiphanes norvegica (M. Sars). Deep-Sea Res. 16:141-156.
Raymont, J. E. G. , R. T. Srinivasagam, and J. K. B. Raymont. 1969b.
Biochemical studies on marine zooplankton. VII. Observations on certain
deep sea zooplankton. Int. Revue ges. Hydrobiol. 54:357-365.
Raymont, J. E. G., R. T. Srinivasagam and J. K. B. Raymont. 1971a.
Biochemical studies on marine zooplankton. VIII. Further investigations
on Meganyctiphanes norvegica (M. Sars). Deep-Sea Res. 18:1167-1178.
Raymont, J. E. G., R. T. Srinivasagam and J. K. B. Raymont. 1971b.
Biochemical studies on marine zooplankton. IX. The biochemical composition
of Euphausla superba. J. mar. biol. Ass. U.K. 51:581-588.
Reeve, M. R., J. E. G. Raymont and J. K. B. Raymont. 1970. Seasonal
biochemical composition and energy sources of Sagitta hispida- Mar. Biol.
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Saiki, M., S. Fang and T. Mori. 1959. Studies on the lipid of plankton.
II. Fatty acid composition of lipids from Euphausiacea collected in the
Antarctic and northern Pacific Ocean. Bull. Jap. Soc. Sci. Fish. 24:837-839.
Sequin, G. 1968. Contribution a 1'etude biochimique de Praunus flexuosus
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Shewbart, K. L., W. L. Mies and P. D. Ludwig. 1972. Identification and
quantitative analysis of the amino acids present in protein of the brown
shrimp Penaeus aztecus. Mar. Biol. 16:64-67.
Sidhu, G. S., W. A. Montgomery, G. L. Holloway, A. R. Johnson and D. M.
Walker. 1970. Biochemical composition and nutritive value of krill
(Euphausia superba Dana). J. Sci. Fd. Agric. 21:293-296.
Simpson, J. W., K. Allen and J. Awapara. 1959. Free amino acids in some
aquatic invertebrates. Biol. Bull. Woods Hole 117:371-381.
Sipos, J. C. and R. G. Ackman. 1968. Jellyfish (Cyanea capillata) lipids;
fatty acid composition. J. Fish. Res. Bd., Can. 25:1561-1569.
Srinivasagam, R. T., J. E. G. Raymont, C. F. Moodie and J. K. B. Raymont.
1971. Biochemical studies on marine zooplankton. X. The amino acid compo-
sition of Euphausia superba, Meganyctiphanes norvegica and Neomysis integer.
J. mar. biol. Ass. U.K. 51:917-925.
Suyama, M., K. Nakajima and J. Nonaka. 1965. Studies on the protein and
non-protein nitrogenous constituents of Euphausia. Bull. Jap. Soc. Scient.
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Vinogradov, A. P. 1953. The elementary chemical composition of marine
)rganisms. Mem. Sears Found. Mar. Res. 2:1-647.
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Vinogradova, Z. A. I960. Study of the biochemical composition of Antarctic
krill (Euphausia superba Dana). Dokl. Akad. Nauk SSSR 133:68-682. (N.L.L.
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Vinogradova, Z. A. 1964. Some biochemical aspects of a comparative study
of plankton from the Black Sea, the Sea of Azov and the Caspian Sea.
Okeanologiya 4(2):232-242.
Webb, K. L. and R. E. Johannes. 1967. Studies of the release of dissolved
free amino acids by marine zooplankton. Limnol. Oceanogr. 12:376-382.
Webb, K. L., R. E. Johannes and S. J. Coward. 1971. Effects of salinity and
starvation on release of dissolved free amino acids by Dugesia dorotocephala
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Yamada, M. 1961. Studies on the lipid of plankton. I. The lipid of
Neomysis nakazawaii. J. Japan Oil Chem. Soc. 10:236-239.
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Zollner, N. and K. Kirsch. 1962. Uber die quantitative bestimmung von
lipoiden (mikromethode) mittels der vie len natur lichen lipoiden (alien
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7.1 Total numbers and frequency of occurrence.
Anraku, M. 1975. Microdistribution of marine copepods in a small inlet.
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Brinton, E. 1962. Variable factors affecting the apparent range and esti-
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7.2 Diversity indices
Goodman, D. 1975. The theory of diversity - stability relationships in
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Haedrich, R. L. 1975. Diversity and overlap as measures of environmental
quality. Water Res. 9:945-952.
Hurlbert, S. H. 1971. The nonconcept of species diversity: a critique and
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Lloyd, M. and R. J. Gherlardi. 1964. A table for calculating the "equitabil-
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Peet, R. K. 1975. Relative diversity indices. Ecology 56:496-498.
Sager, P. and A. D. Easier. 1969. Species diversity in lacustrine phyto-
plankton. I. The components of the index of diversity from Shannon's
formula. Amer. Natur. 103:51-59.
Sanders, H. L. 1968. Marine benthic diversity: a comparative study. Am.
Natur. 102:243-282.
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51
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-78-026
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
GUIDELINES FOR ZOOPLANKTON SAMPLING IN QUANTITATIVE
BASELINE AND MONITORING PROGRAMS
5. REPORT DATE
February 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Fred Jacobs and George C. Grant
8. PERFORMING ORGANIZATION REPORT NO
Special Scientific Report
No. 83
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Planktology
Virginia Institute of Marine Science
Gloucester Point, VA 23062
10. PROGRAM ELEMENT NO.
1 BA 025
11. CONTRACT/GRANT NO.
EPA-R-804147010
12. SPONSORING AGENCY NAME AND ADDRESS
Corvallis Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Methods applicable to zooplankton sampling and analysis in quantitative baseline
and monitoring surveys are evaluated and summarized. Specific recommendations by
managers must take into account characteristics of the water mass under investi-
gation, the abundance of contained zooplankton and phytoplankton populations and
the objectives of the study. Realistic planning and development must also consider
available monetary and manpower resources.
This report was submitted in fulfillment of Contract No. R804147010 by the Virginia
Institute of Marine Science under the sponsorship of the U.S. Environmental Pro-
tection Agency. This report covers a period from 24 Nov 75, to 31 May 77, and
work was completed as of 25 Feb 77.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATl F-'ield/Group
Marine biology
Sampling
Water pollution
Zooplankton
08/A
8. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report I
unclassified
21. NO. OF PAGES
60
20. SECURITY CLASS (Thispage)
unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
52
U.S. GOVERNMENT PRINTING OFflCEi 1978—799-946/86 REGION 10
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