10-2 BIOLOGICAL EXAMINATION (10000)

10200 PLANKTON*

10200 A. Introduction

The term “plankton” refers to those aquatic forms having little or logical aspects of water quality (such as pH, color, taste, oxygen
no resistance to currents and living free-floating and suspended in concentration, and odor), and in a very practical sense, they are a
natural waters. Planktonic plants, “phytoplankton,” and planktonic part of water quality. Certain taxa often are useful in determining
animals, “zooplankton,” are covered in this section. The phyto- the origin or recent history of a given water mass. Because of their
plankton (microscopic algae) occur as unicellular, colonial, or fila- transient nature, and often patchy distribution, however, the utility
mentous forms. Most of these are photosynthetic and are grazed of plankters as water quality indicators may be limited. Information
upon by zooplankton and other aquatic organisms. Other organisms on plankton as indicators is interpreted best in conjunction with
occurring in the same environment are dealt with elsewhere: zoo- concurrently collected, physicochemical and other biological data.
sporic fungi in Section 9610F; aquatic hyphomycetes in Section Algal blooms frequently occur during the summer and early
9610G; and bacteria in Part 9000. The zooplankton in fresh water fall months. These are most common in eutrophic waters and
comprise principally protozoans, rotifers, cladocerans, and cope- may be associated with periods of reduced oxygen concentra-
pods; a greater variety of organisms occurs in marine waters. tions in the water, unpleasant ordors, and, at times, fish deaths.
There are also cyanobacteria and dinoflagellate taxa (among
1. Significance others) that produce toxins that may be harmful to fish, and other
animals in contact with waters containing the toxins. Several of
Plankton, particularly phytoplankton, have long been used as these toxins have been found in seafood and drinking water, and
indicators of water quality.1– 4 Some species flourish in highly have produced illness and fatalities in humans.20 –22
eutrophic waters while others are very sensitive to organic and/or
2. References
chemical wastes. Some species develop noxious blooms, some-
times creating offensive tastes and odors5 or anoxic or toxic
1. PALMER, C.M. 1969. A composite rating of algae tolerating organic
conditions resulting in animal deaths or human illness.6 The pollution. J. Phycol. 5:78.
species assemblage of phytoplankton and zooplankton may be 2. PALMER, C.M. 1963. The effect of pollution on river algae. Bull. N.Y.
useful in assessing water quality.7–13 Plankton also may be used Acad. Sci. 108:389.
as indicators of relative treatment efficiencies of water treatment 3. RAWSON, D.S. 1956. Algal indicators of trophic lake types. Limnol.
plants and the probability of groundwater sources under the Oceanogr. 1:18.
direct influence of surface water.14 –19 4. STOERMER, E.F. & J.J. YANG. 1969. Plankton Diatom Assemblages
Because of their short life cycles, plankters respond quickly to in Lake Michigan. Spec. Rep. No. 47, Great Lakes Research Div.,
environmental changes, and hence their standing crop and species Univ. Michigan, Ann Arbor.
composition are more likely to indicate the quality of the water mass 5. PRESCOTT, G.W. 1968. The Algae: A Review. Houghton Mifflin Co.,
Boston, Mass.
in which they are found. They strongly influence certain nonbio-
6. CARMICHAEL, W., ed. 1981. The Water Environment, Algal Toxins
and Health. Plenum Press, New York, N.Y.
* Approved by Standard Methods Committee, 2001.
7. GANNON, J.E. & R.S. STEMBERGER. 1978. Zooplankton (especially
Joint Task Group: Harold G. Marshall (chair), Robert R. Bidigare, Ernst M. Davis, crustaceans and rotifers) as indicators of water quality. Trans. Amer.
Robert A. Sweeney. Microsc. Soc. 97:16.
PLANKTON (10200)/Sample Collection 10-3

8. TOMAS, C.R., ed. 1997. Identifying Marine Phytoplankton. Aca- 16. CLANCY, J.L. 1992. Interpretation of microscopic particulate analy-
demic Press, Harcourt Brace & Co., San Diego, Calif. sis data—A water quality approach. In Proc. American Water
9. PLATT, T. & W.K.W. LI, eds. 1986. Photosynthetic Picoplankton. Works Association Water Quality Technology Conference, To-
Canadian Bull. Fish. Aquatic Sci. 214, Dep. Fisheries and Oceans, ronto, Canada, p. 1831. American Water Works Assoc., Denver,
Ottawa, Ont. Colo.
10. STEVENSON, R.J. & K.D. WHITE. 1995. A comparison of natural and 17. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1996. Microscopic Par-
human determinants of phytoplankton communities in the Kentucky ticulate Analysis (MPA) for Filtration Plant Optimization. EPA
River basin, U.S.A. Hydrobiologia 297:201. 910/R-96-001, U.S. Environmental Protection Agency, Seattle,
11. LANGE-BERTALOT, H. 1979. Pollution tolerance of diatoms as crite- Wash.
rion for water quality estimation. Nova Hedwigia 64:285. 18. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1992. Consensus Method
12. DIXIT, S.S. & J.P. SMOL. 1994. Diatoms as indicators in environ-
for Determining Groundwaters Under the Direct Influence of Sur-
mental monitoring and assessment program-surface waters (EMAP-
face Water Using Microscopic Particulate Analysis (MPA). EPA
SW). Environ. Monitor. Assess. 31:275.
910/9-92-029, U.S. Environmental Protection Agency, Port Or-
13. STOERMER, E.F. & J.P. SMOL, eds. 1999. The Diatoms: Applications
chard, Wash.
for the Environmental and Earth Sciences. Cambridge University
Press, Cambridge, U.K. 19. HANCOCK, C.M., J.V. WARD, K.W. HANCOCK, P.T. KLONICKI & G.D.
14. PORTER, S.D., T.F. CUFFNEY, M.E. GURTZ & M.R. MEADOR. 1993. STURBAUM. 1996. Assessing plant performance using MPA. J. Amer.
Methods for collecting algae as part of the National Water-Quality Water Works Assoc. 88:24.
Assessment Program. USGS Open-file rept. 93-409, U.S. Geologi- 20. HALLEGRAEFF, G.M. 1991. Aquaculturists’ Guide to Harmful Aus-
cal Survey, Raleigh, N.C. tralian Microalgae. Fishing Industry Training Board of Tasmania,
15. CLARK, S.C., M.L. PRICE, J. FLUGUM & R. ROBERSON. 1993. Ground- Hobart, Tasmania, Australia.
water under the direct influence of surface water: it is not always 21. FALCONER, I.R., ed. 1993. Algal Toxins in Seafood and Drinking
black or white. In Proc. American Water Works Association Water Water. Academic Press, Harcourt Brace & Co., San Diego, Calif.
Quality Technology Conf., Miami, Fla., p. 703. American Water 22. WATANABE, M.F., K. HARADA, W.W. CARMICHAEL & H. FUJIKI, eds.
Works Assoc., Denver, Colo. 1995. Toxic Microcystis. CRC Press, Boca Raton, Fla.

10200 B. Sample Collection

1. General Considerations Because water of rivers and streams usually is well mixed
vertically, subsurface sampling, i.e., the upper meter or a com-
The frequency and location of sampling is dictated by the posite of two or more strata, often is adequate for collection of
purpose of the study.1 Locate sampling stations as near as a representative sample. There may be problems caused by
possible to those selected for chemical and bacteriological stratification due to thermal discharges or mixing of warmer or
sampling to insure maximum correlation of findings. Estab- colder waters from tributaries and reservoirs. Always sample in
lish a sufficient number of stations in as many locations as the main channel of a river and avoid sloughs, inlets, or back-
necessary to define adequately the kinds and quantities of water areas that reflect local habitats rather than river conditions.
plankton in the waters studied. The physical nature of the In rivers that are mixed vertically and horizontally, measure
water (standing, flowing, or tidal) will influence greatly the plankton populations by examining periodic samples collected at
selection of sampling stations. The use of sampling sites midstream 0.5 to 1 m below the surface.
selected by previous investigators usually will assure the If it can be determined or correctly assumed that the plankton
availability of historical data that will lead to a better under- distribution is uniform and normal, use a scheme of random
standing of current results and provide continuity in the study sampling to accomodate statistical testing. Include both random
of an area. selection of sampling sites and transects as well as the random
In stream and river work, locate stations upstream and down- collection of samples at each selected site. On the other hand, if
stream from suspected pollution sources and major tributary it is known or assumed that plankton distribution is variable or
streams and at appropriate intervals throughout the reach under patchy, include additional sampling sites, collect composite sam-
investigation. If possible, locate stations on both sides of the ples, and increase sample replication. Use appropriate statistical
river because lateral mixing of river water may not occur for tests to determine population variability.
great distances downstream. In a similar manner, investigate In sampling a lake or reservoir use a grid network or transect
tributary streams suspected of being polluted but take care in the lines in combination with random procedures. Take a sufficient
interpretation of data from a small stream because much of the number of samples to make the data meaningful. Sample a
plankton may be periphytic in origin, arising from scouring of circular lake basin at strategic points along a minimum of two
natural substrates by the flowing water. Plankton contributions perpendicular transects extending from shore to shore; include
from adjacent lakes, reservoirs, and backwater areas, as well as the deepest point in the basin. Sample a long, narrow basin at
soil organisms carried into the stream by runoff, also can influ- several points along a minimum of three regularly spaced par-
ence data interpretation. The depth from which water is dis- allel transects that are perpendicular to the long axis of the basin,
charged from upstream stratified reservoirs also can affect the with the first near the inlet and the last near the outlet. Sample a
nature of the plankton. large bay along several parallel transects originating near shore
10-4 BIOLOGICAL EXAMINATION (10000)

and extending to the lake proper. Because many samples are over at least two complete tidal cycles. Once a characteristic
required to appraise completely the plankton assemblage, it may pattern is recognized the sampling routine may be modified.
be necessary to restrict sampling to strategic points, such as the A useful series of monographs on oceanographic methodology
vicinity of water intakes and discharges, constrictions within the has been published.4 –7 Representative taxonomic references for
water body, and major bays that may influence the main basin. estuarine and marine phytoplankton include diatoms,8 –11
In lakes, reservoirs, and estuaries where plankton populations dinoflagellates,12–14 coccolithophores,15 and cyanophyceae16
can vary with depth, collect samples from all major depth zones (cyanobacteria).
or water masses. The sampling depths will be determined by the
water depth at the station, the depth of the thermocline or an 2. Sampling Procedures
isohaline, or other factors. In shallow areas of 2 to 3 m depth,
subsurface samples collected at 0.5 to 1 m may be adequate. In Once sampling locations, depths, and frequency have been
deeper areas, collect samples at regular depth intervals. In estu- determined, prepare for field sampling. Label sample containers
aries sample above and below the pyncocline. Depth intervals for with sufficient information to avoid confusion or error. On the
sampling vary for estuaries of different sizes and depths, but use label indicate date, cruise number, sampling station, study area
depths representative of the vertical range. Composite sampling (river, lake, reservoir), type of sample, and depth. Use water-
above and below the pyncocline often is used. In marine sam- proof labels. When possible, enclose collection vessels in a
pling, the intent and scope of the study will determine the protective container to avoid breakage. If samples are to be
collection extent. preserved immediately after collection, add preservative to con-
Over the continental shelf, take samples at stations approxi- tainer before sampling. Sample size depends on type and number
mately equidistant from the shore seaward. Take a vertical series of determinations to be made; the number of replicates depends
from surface to near bottom at each station, gradually adding on statistical design of the study and statistical analyses selected
more stations across the shelf. It is important to sample the entire for data interpretation. Always design a study around an objec-
vertical range over a continental shelf. Benthic grab samples may tive with a statistical approach rather than fit statistical analyses
be taken to collect dormant resting cells or cysts. Beyond the to data already collected.
shelf in pelagic waters, sample in the photic zone from the In a field record book note sample location, depth, type, time,
surface to the thermocline for phytoplankton and to deeper meteorological conditions, turbidity, water temperature, salinity,
depths for zooplankton. Sampling depths vary, but often are at and other significant observations. Engineer’s field notebooks
10- to 25-m intervals above the thermocline, then at 100- to with waterproof paper are very suitable. Field data are invaluable
200-m intervals below the thermocline to 1000 m, and thereafter when analytical results are interpreted and often help to explain
at 500- to 1000-m intervals. unusual changes caused by the variable character of the aquatic
Samples usually are referred to as “surface” or “depth” (sub- environment. Collect coincident samples for chemical analyses
surface) samples. The latter are samples taken from some stated to help define environmental variations having a potential effect
depth, whereas surface samples may be interpreted as samples on plankton.
collected as near the water surface as possible. A “skimmed” a. Phytoplankton: In oligotrophic waters or where phytoplank-
sample of the surface film plankton (neuston)2 can be revealing; ton densities are expected to be low collect a sample of up to 6
however, ordinarily do not include a disproportionate quantity of L. For richer, eutrophic waters collect a sample of 0.5 to 1 L.
surface film in a surface sample because a neustonic flora3 as Because of their small size, nannoplankton and picoplankton
well as plankton often are trapped on top or at the surface film can pass through collection nets, making nets unsuitable for most
together with pollen, dust, and other detritus. Various methods phytoplankton sampling.
have been used for sampling surface organisms. For qualitative and quantitative evaluations collect whole (un-
Sampling frequency depends on the intent of the study as well filtered and unstrained) water samples with a water collection
as the range of seasonal fluctuations, the immediate meteorolog- bottle consisting of a cylindrical tube with stoppers at each end
ical conditions, adequacy of equipment, and availability of per- and a closing device. Lower the open sampler to the desired
sonnel. Select a sampling frequency at some interval shorter than depth and close by dropping a weight, called a messenger, which
community turnover time. This requires consideration of life- slides down the supporting wire or cord and trips the closing
cycle length, competition, predation, flushing, and current dis- mechanism. If possible, obtain composite samples from several
placement. Frequent plankton sampling is desirable because of depths or pool samples from one depth from several casts. The
normal temporal variability and migratory character of the most commonly used samplers that operate on this principle are
plankton community. Daily vertical migrations occur in response the Kemmerer,17 Van Dorn18 (Figure 10200:1), Niskin, and
to sunlight, and random horizontal migrations or drifts are pro- Nansen samplers.
duced by winds, shifting currents, and tides. Ideally, collect daily Because these samplers collect whole water samples, all size
samples and, when possible, sample at different times during the classes of phytoplankton are collected. Different size categories
day and at different depths. When this is not possible, weekly, of phytoplankton can be separated by subsequently filtering
biweekly, monthly, or even quarterly sampling still may be these whole water samples through netting of the appropriate
useful for determining major population changes. mesh size. Select appropriate mesh sizes for concentrating the
In river, stream, and estuarine regions subject to tidal influ- various size categories of phytoplankton typical of the aquatic
ence, expect fluctuations in plankton composition over a tidal system under study.19,20
cycle. A typical sampling pattern at a station within an estuary The Van Dorn usually is the preferred sampler for standing
includes a vertical series of samples taken from the surface, crop, primary productivity, and other quantitative determinations
across the pyncocline, to near bottom, collected at 3-h intervals, because its design offers no inhibition to free flow of water
PLANKTON (10200)/Sample Collection 10-5

For greater speed of collection and to obtain large, accurately

measured quantities of organisms, use a pump. Diaphragm and
peristaltic pumps are less damaging to organisms than centrifu-
gal pumps.24 Centrifugal pump impellers can damage organisms
as can passage through the hose.25 Lower a weighted hose,
attached to a suction pump, to the desired depth, and pump water
to the surface. The pump is advantageous because it supplies a
homogeneous sample from a given depth or an integrated sample
from the surface to a particular depth. If a centrifugal pump is
used, draw samples from the line before they reach the impeller.
For samples to be analyzed for organochlorine compounds use
TFE tubing.
To examine live samples fill containers partially and store in a
refrigerator or ice chest in the dark, or preferably, hold at
ambient temperature. Examine specimens promptly after collec-
tion.
If it is impossible to examine living material or if phytoplank-
ton are to be counted later, preserve the sample. For a sample
that will be preserved, fill the container completely, leaving
sufficient air space to permit mixing the sample by shaking. The
most suitable phytoplankton preservative is Lugol’s solution,
which can be used for most forms including the naked flagel-
lates. Unfortunately, acidic Lugol’s solution (or formalin) dis-
solves the coccoliths of Coccolithophores, which are common in
estuarine and marine waters.
Lugol’s solution: To preserve samples with Lugol’s solution
add 0.3 mL Lugol’s solution to 100 mL sample and store in the
dark. For long-term storage add 0.7 mL Lugol’s solution per 100
mL sample and buffered formaldehyde to a minimum of 2.5%
final concentration after 1 h. Prepare Lugol’s solution by dis-
solving 20 g potassium iodide (KI) and 10 g iodine crystals in
200 mL distilled water containing 20 mL glacial acetic acid.26
Utermohl’s27 modification of Lugol’s solution results in a neutral
or slightly alkaline solution. Prepare modified Lugol’s solution
by dissolving 10 g KI and 5 g iodine crystals in 20 mL distilled
water, then adding 50 mL distilled water in which 5 g anhydrous
sodium acetate has been dissolved. This allows preservation of
Coccolithophores, but would be less effective for other flagel-
lates.
Figure 10200:1. Structural features of common water samplers, Kem- Other acceptable preservatives are:
merer (left) and Van Dorn (right). Formalin: To preserve samples with formalin, add 40 mL
buffered formalin (20 g sodium borate, Na2B2O4, ⫹ 1 L 37%
formaldehyde) to 1 L of sample immediately after collection. In
through the cylinder. In deep-water situations, the Niskin bottle estuarine and marine collections, adjust pH to at least 7.5 with
is preferred. It has the same design as the Van Dorn sampler sodium borate for samples containing Coccolithophores.
except that the Niskin sampler can be cast in a series on a single Merthiolate: To preserve samples with merthiolate add 36 mL
line for simultaneous sampling at multiple depths with the use of merthiolate solution to 1 L of sample and store in the dark.
auxiliary messengers. Because the triggering devices of these Prepare merthiolate solution by dissolving 1.0 g merthiolate,
samplers are very sensitive, avoid rough handling. Always lower 1.5 g sodium borate, and 1.0 mL Lugol’s solution in 1 L distilled
the sampler into the water; do not drop. Kemmerer and Van Dorn water. Merthiolate-preserved samples are not sterile, but can be
samplers have capacities of 0.5 L or more. Polyethylene or kept effectively for 1 year, after which time formalin must be
polyvinyl chloride sampling devices are preferred to metal sam- added.28
plers because the latter liberate metallic ions that may contam- “M3” fixative: Prepare by dissolving 5 g KI, 10 g iodine, 50
inate the sample. Use polyethylene or glass sample storage mL glacial acetic acid, and 250 mL formalin in 1 L distilled
bottles. Metallic ion contamination can lead to significant errors water (dissolve the iodide in a small quantity of water to aid in
when algal assays or productivity measurements are made. solution of the iodine). Add 20 mL fixative to 1 L sample and
For shallow waters use the Jenkins surface mud sampler,21 one store in the dark.
of the bottle samplers modified so that it is held horizontally,22 Glutaraldehyde: Preserve samples by adding neutralized glu-
or an appropriate bacteriological sampler.23 taraldehyde to yield a final concentration of 1 to 2%.
10-6 BIOLOGICAL EXAMINATION (10000)

Other commonly used preservatives include 95% alcohol, and

6-3-1 preservative, (6 parts water, 3 parts 95% alcohol, and 1
part formalin). Use equal volumes of preservative and sample.
To retain color in preserved plankton, store samples in the
dark or add 1 mL saturated copper sulfate (CuSO4) solution/L.
Most preservatives distort and disrupt certain cells,29,30 espe-
cially those of delicate forms such as Euglena, Cryptomonas,
Synura, Chromulina, and Mallamonas. Lugol’s iodine solution
usually is least damaging for these phytoflagellates. To become
familiar with live specimens and preservation-caused distortions,
use reference collection from biological supply houses or consult
experienced co-workers.
b. Zooplankton: The choice of sampler depends on the type of
zooplankton, the kind of study (distribution, productivity, etc.)
and the body of water being investigated. Zooplankton popula-
tions invariably are distributed in a patchy way, making both
sampling and data interpretation difficult.
For collecting microzooplankton (20 to 200 ␮m) such as proto-
zoa, rotifers, and immature microcrustacea, use the bottle samplers
described for phytoplankton. The small zooplankters usually are
sufficiently abundant to yield adequate samples in 5- to 10-L bot-
tles; however, composite samples over depth and time are recom-
mended. Water bottle samplers are suitable especially for discrete-
depth samples. If depth-integrated samples are desired, use pumps
or nets. The larger and more robust microzooplankters (e.g., loricate
forms and crustacea) may be concentrated by passing the whole
water through a 20-␮m mesh net. If quantitative estimates of other
nonloricate, delicate forms are required, do not screen. Fix 0.5 to 5
L of whole water for enumeration of these forms.
Bottle samplers usually are unsuitable for collecting larger
zooplankton, such as mature microcrustacea, that, unlike the
smaller forms, are much less numerous and are sufficiently agile
to avoid capture. Although comparatively large water volumes,
and consequently adequate numbers of microcrustacea, can be
sampled with a pump, avoidance by larger, more agile zooplank-
ters at the pump head can cause sampling error. Consequently,
larger trap samplers or nets are the preferred collection methods.
The Juday trap31 operates on the same principle as the water Figure 10200:2. The Schindler-Patalas plankton trap.
bottle samplers but is generally larger (10 L). The larger size
makes the Juday trap more suitable for collecting zooplankters,
especially larger copepods. However, it is awkward to use and its particular needs of the study.33,34 Type of netting and mesh size
10-L capacity is inadequate for oligotrophic lakes or other water determine filtration efficiency, clogging tendencies, velocity,
bodies with few zooplankters. Because it is constructed of metal drag, and the condition of the sample after collection. Silk,
it is unsuited if heavy metals analyses are required. formerly the common mesh material in plankton nets, is not
The Schindler-Patalas trap32 (Figure 10200:2) usually is preferred recommended because of shrinkage of mesh openings and rot-
to the Juday trap because it is constructed of clear acrylic plastic and ting with age. Nylon monofilament mesh is preferred because of
is transparent. It can be lowered into the water with minimal its mesh size accuracy and durability. Nylon nets of different
disturbance and is suitable for collecting larger zooplankters. Mod- mesh sizes still are labelled by the silk rating system: character-
els of 10- to 12-L capacity are available but the 30-L size is istics of commonly used nylon plankton nets are listed in Table
preferred. It has no mechanical closing mechanism and thus is 10200:I. Finer mesh sizes clog more readily than coarser mesh;
convenient for cold-weather sampling when mechanical devices a compromise must be made between mesh size small enough to
tend to malfunction. Like the Juday trap, it can be fitted with nets of retain desired organisms effectively and a size large enough to
various mesh sizes, but the No. 20 mesh net is used most often. preclude a serious clogging problem. If clogging occurs, reduce
Plankton nets are preferred to bottles and traps for sampling its effects by decreasing the length of tow.
where plankters are few or where only qualitative data or a large The maximum volume, VM, of water that can be filtered through
biomass is needed for analysis. Because they were designed a net during a vertical tow can be estimated with the formula,
originally for qualitative sampling, modifications are required for
quantitative work. V M ⫽ ␲r 2d
The mesh size, type of material, orifice size, length, hauling
method, type of tow, and volume sampled will depend on the where:
PLANKTON (10200)/Sample Collection 10-7

TABLE 10200:I. CHARACTERISTICS OF COMMONLY USED PLANKTON NETS

Size of Approximate
Aperture Open Area
Silk No. ␮m % Classification

000 1024 58 Largest zooplankton and ichthyoplankton

00 752 54 Larger zooplankton and ichthyoplankton
0 569 50 Large zooplankton and ichthyoplankton
2 366 46 Large microcrustacea
6 239 44 Microcrustacea
10 158 45 Microcrustacea and most rotifers
20 76 45 Net phyto- and zooplankton
25 64 33 Nannoplankton

r ⫽ radius of net orifice and

d ⫽ depth to which net is lowered.

This volume is a maximum because clogging of the net’s

meshes by phytoplankton and other particles and, for fine net-
ting, even the netting itself can cause some water to be diverted
from the net’s path.35,36 Keep net towing distance as short as
practical to alleviate clogging. If the net has a pronounced green
or brown color after towing, clogging probably has occurred.
To estimate sampling volume, VA, mount a calibrated flow
meter midway between the net rims and mouth center (the meter
is mounted off-center to avoid flow reduction associated with the
towing bridle).37 Equip meter with lock mechanisms to prevent
it turning in reverse or while in air. Record flow-meter readings
before and after collecting sample. Calculate filtration efficiency,
E, from:

E ⫽ V A /V M

If E is less than about 0.8, substantial clogging has occurred.

Take steps to increase efficiency. Clogging not only decreases
the volume filtered, but also leads to biased samples because
filtration efficiency is nonuniform during the tow.34
Various types of plankton nets are shown in Figure 10200:3.
Simple conical nets have been used for many years with little
modification in design or improvement in accuracy. Their major
source of error is that the filtration characteristics of conical nets
usually are unknown. Filtration efficiency in No. 20 mesh cone
nets ranges from 40 to 77%. To improve efficiency, place a
porous cylinder collar or nonporous truncated cone in front of
the conical portion of the net. The Juday net exemplifies a
commonly used net with a truncated cone. For good filtration
characteristics the ratio of filtering area of net to orifice area
should be at least 3:1. Bridles attaching the net to the towing line
also adversely influence filtration efficiency and increase turbu-
lence in front of the net, thereby increasing the potential for net
avoidance by larger zooplankters. The tandem, Bongo net design
(Figure 10200:3) reduces these influences and permits duplicate
samples to be collected simultaneously.
Figure 10200:3. Examples of commonly used plankton sampling nets.
Three types of tows are used: vertical, horizontal, and oblique.
(A) Simple conical tow-net; A—rigged for vertical tows;
Vertical tows are preferred to obtain an integrated water column
A1—for oblique or horizontal tows; (B) Wisconsin (Birge)
sample. To make a vertical tow, lower the weighted net to a tow-net with truncated cone to improve filtration efficiency;
given depth, then raise vertically at an even speed of 0.5 m/s. (C) Bongo net, can be fitted with flow meters and opening/
In small water bodies haul the net hand over hand with a closing mechanisms; (D) Wisconsin net fitted with messen-
steady, unhurried motion approximating the speed of 0.5 m/s. In ger-activated closing mechanism, D— open, D1— closed;
large bodies where long net hauls and vessel drifting are ex- (E) Free-fall net, E— open, E1— closed.
10-8 BIOLOGICAL EXAMINATION (10000)

horizontal tows use a boat equipped as above and determine

sampler depth as above. Lower sampler to preselected depth,
open, tow at that depth for 5 to 10 min, then close and raise it.
A variety of zooplankton sampling methods can be used in
flowing water. The method of choice depends largely on flow
velocity. Properly weighted bottles, traps and pump hoses, and
nets can be used in medium- to slow-flowing waters. In turbu-
lent, well-mixed waters, collect surface water by bucket and
filter it through the appropriate mesh size. Select sample size
based on concentration of zooplankters.
Give plankton nets proper care and maintenance. Do not let
particulate matter dry on the net because it can significantly
reduce size of mesh apertures and increase frequency of clog-
ging. Wash net thoroughly with water after each use. Periodi-
cally clean with a warm soap solution. Because nylon net ma-
terial is susceptible to deterioration from abrasion and sunlight,
guard against unnecessary wear and store in the dark.
Traps and nets do not work well in shallow areas with growths
of aquatic vegetation. To obtain an integrated sample for the
entire water column in such areas, use a length of light-weight
rubber or polyethylene tubing with netting attached over one end
and a rope on the other.39 Attach netting by tape or rubber bands
that will stay in place in water, but can be removed easily after
sampling. Use tubing of 5- to 10-cm diam and long enough to
reach from the surface to the bottom. Lower the open end (the
end with the rope attached) until it almost touches the bottom.
Then pull this end up using the rope and keep the covered end
above the water surface. When the open end is out of the water,
let the end with the netting fall back into the water, pull the
tubing into the boat, open end first, and let the water in the tube
drain out through the netting. When the zooplankton has been
Figure 10200:4. Examples of commonly used high-speed zooplankton concentrated in a small volume, just above the netting, remove
samplers. (A) Clarke-Bumpus sampler; (B) Miller sam- the netting over a container and catch the concentrated sample.
pler; (C) Hardy plankton indicator; (D) Hardy continuous
Wash netting and end of tubing into the container to assure that
plankton recorder; (E) Issacs-Kidd mid-water trawl; (F)
Gulf V sampler; (G) Tucker trawl, G1—side view, G2—
all the zooplankton is collected. This method is not limited to
front view open and closed. areas with aquatic vegetation. It provides an excellent method of
obtaining an integrated sample from any shallow area. In stand-
ing waters, collect tow samples by filtering 1 to 5 m3 of water.
Preserve zooplankton samples with 70% ethanol or 5% buff-
pected, use a davit, meter wheel, angle indicator, and winch. ered formalin. Ethanol preservative is preferred for materials to
Attach a 3- to 5-kg weight to hold the net down. Determine depth be stained in permanent mounts or stored. Formalin may be used
of the net by multiplying the length of the extended wire by the for the first 48 h of preservation with subsequent transfer to 70%
cosine of the wire’s angle with the vertical direction. Maintain ethanol. Formalin preservative may cause distortion of pleomor-
wire angle as close to the vertical as possible by controlling the phic forms such as protozoans and rotifers. Make formalin in
boat’s speed null against the wind drift, or wherever feasible, do sucrose-saturated water to minimize carapace distortion and loss
vertical hauls from an anchored boat. of eggs in crustaceans, especially cladocerans.40 Bouin’s fixative
Vertical and oblique tows collect a composite sample, whereas produces reasonable results for soft-bodied microzooplankton.41
horizontal tows collect a sample at a discrete depth. Oblique This fixative is picric acid saturated in calcium carbonate-buff-
tows usually are preferred over vertical tows in shallow water or ered formaldehyde containing 5% (v/v) acetic acid. Dilute
wherever a longer net tow is required. For oblique tows, lower Bouin’s fixative 1:19 with the sample. Because rapid fixation is
the net or sampler to some predetermined depth and then raise at necessary, pour the sample onto the fixative or inject fixative
a constant rate as the boat moves forward. Oblique tows do not rapidly into the sample.
necessarily sample a true angle from the bottom to the surface. Use a narcotizing agent such as carbonated water, menthol-
Under best conditions the pattern is somewhat sigmoid due to saturated water, or neosynephrine to prevent or reduce contrac-
boat acceleration and slack in the tow line. tion or distortion of organisms, especially rotifers, cladocerans,
Horizontal tows usually are used to obtain depth distribution and many marine invertebrates.42,43 Adding a few drops of
information on zooplankton. Although a variety of horizontal detergent prevents clumping of preserved organisms. Preserve
samplers is available (see Figure 10200:4), use the Clarke- samples as soon as most animal movement has ceased, usually
Bumpus sampler38 for quantitative collection of zooplankton within a half hour of narcotization. To prevent evaporation, add
because of its built-in flowmeter and opening-closing device. For 5% glycerin to the concentrated sample. In turbid samples,
PLANKTON (10200)/Sample Collection 10-9

differentiate animal and detrital material by adding 0.04% rose 21. MORTIMER, C.H. 1942. The exchange of dissolved substances be-
bengal stain, which intensely stains the carapace (shell) of zoo- tween mud and water in lakes. J. Ecol. 30:147.
plankters and is a good general cytoplasmic stain. 22. VOLLENWEIDER, R.A. 1969. A Manual on Methods for Measuring
Primary Production in Aquatic Environments. IBP Handbook No.
12. Blackwell Scientific Publ., Oxford, England.
3. References 23. GELDREICH, E.E., H.D. NASH, D.F. SPINO & D.J. REASONER. 1980.
Bacterial dynamics in a water supply reservoir: a case study.
1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1982. Handbook for J. Amer. Water Works Assoc. 72:31.
Sampling and Sample Preservation of Water and Wastewater. EPA- 24. BEERS, J.R. 1978. Pump sampling. In A. Sournia, ed. Phytoplankton
600/4-82-029. Manual. United Nations Educational, Scientific and Cultural Org.,
2. PARKER, B.C. & R.F. HATCHER. 1974. Enrichment of surface fresh- Paris.
water microlayers with algae. J. Phycol. 10:185. 25. EXTON, R.J., W.M. HOUGHTON, W. ESAIAS, L.W. HAAS & D. HAY-
3. TAGUCHI, S. & K. NAKAJIMA. 1971. Plankton and seston in the sea WARD. 1983. Spectral differences and temporal stability of phyco-
surface of three inlets of Japan. Bull. Plankton Soc. Japan 18:20. erythrin fluorescence in estuaries and coastal waters due to the
4. UNITED NATIONS EDUCATIONAL, SCIENTIFIC AND CULTURAL ORGANIZA- domination of labile cryptophytes and stable cyanobacteria. Limnol.
TION. 1966. Determination of Photosynthetic Pigments in Sea-water. Oceanogr. 28:1225.
Monogr. Oceanogr. Methodol. No. 1. United Nations Educational, 26. EDMONDSON, W.T., ed. 1959. Freshwater Biology, 2nd ed. John
Scientific & Cultural Org., Paris. Wiley & Sons, New York, N.Y.
5. UNITED NATIONS EDUCATIONAL, SCIENTIFIC AND CULTURAL ORGANIZA- 27. UTERMÖHL, H. 1958. Zur Vervollkommung der quantitativen Phy-
TION. 1968. Zooplankton Sampling. Monogr. Oceanogr. Methodol.
toplankton-Methodik. Int. Ver. Theoret. Angewand. Limnol., Com-
No. 2. United Nations Educational, Scientific & Cultural Org., mun. No. 9.
Paris. 28. WEBER, C.I. 1968. The preservation of phytoplankton grab samples.
6. UNITED NATIONS EDUCATIONAL, SCIENTIFIC AND CULTURAL ORGANIZA- Trans. Amer. Microsc. Soc. 87:70.
TION. 1973. A Guide to the Measurement of Marine Primary Pro-
29. PAERL, H.W. 1984. An evaluation of freeze fixation as a phytoplank-
duction under Some Special Conditions. Monogr. Oceanogr. Meth- ton preservation method for microautoradiography. Limnol. Ocean-
odol. No. 3. United Nations Educational, Scientific & Cultural Org., ogr. 29:417.
Paris.
30. SILVER, M.W. & P.J. DAVOLL. 1978. Loss of 14C activity after
7. SOURNIA, A., ed. 1978. Phytoplankton Manual. Monogr. Oceanogr.
chemical fixation of phytoplankton: Error source for autoradiogra-
Methodol. No. 6. United Nations Educational, Scientific & Cultural
phy and other productivity measurements. Limnol. Oceanogr. 23:
Org., Paris.
362.
8. CUPP, E.E. 1943. Marine plankton diatoms of the west coast of
31. JUDAY, C. 1916. Limnological apparatus. Trans. Wis. Acad. Sci. 18:566.
North America. Bull. Scripps Inst. Oceanogr. 5:1.
32. SCHINDLER, D.W. 1969. Two useful devices for vertical plankton and
9. HUSTEDT, F. 1927– 66. Die Kieselalgen Deutschlands, Österreichs
water sampling. J. Fish. Res. Board Can. 26:1948.
und der Schweiz mit Berucksichtigung der Übrigen Lander Europas
33. SCHWOERBEL, J. 1970. Methods of Hydrobiology. Pergamon Press,
Sowie der Angrenzenden Meeresgebiete. In L. Rabenhorst, Kryp-
Toronto, Ont.
togamen-Flora. Vol. 7: Teil 1 (1927–30); Teil 2 (1931–59); Teil 3
34. TRANTER, D.J., ed. 1980. Reviews on Zooplankton Sampling Meth-
(1961– 66). Akademie Verlag, Leipzig, Germany.
10. LEBOUR, M.V. 1930. The Planktonic Diatoms of Northern Seas. Ray ods. United Nations Educational, Scientific & Cultural Org., Swit-
Soc., London. zerland.
11. HENDEY, N.I. 1964. An introductory account of the smaller algae of 35. GANNON, J.E. 1980. Towards improving the use of zooplankton in
British coastal waters, V. Bacillariophyceae (Diatoms). Fish. Invest. water quality surveillance of the St. Lawrence Great Lakes. Proc.
Min. Agr. Fish. Food (G.B.), Ser. IV:1. 1st Biol. Surveillance Symp., 22nd Conf. Great Lakes Research
12. DODGE, J.D. 1975. The prorocentrales (Dinophyceae), II. Revision Can. Tech. Rep. Fish. Aquat. Sci. 976, p. 87.
of the taxonomy within the genus Prorocentrum. Bot. Limnol. Soc. 36. ROBERTSON, A. 1968. Abundance, distribution, and biology of plank-
71:103. ton in Lake Michigan with the addition of a Research Ships of
13. LEBOUR, M.V. 1925. The Dinoflagellates of Northern Seas. Marine Opportunity project. Spec. Rep. No. 35, Great Lakes Research Div.,
Biological Assoc. United Kingdom, Plymouth. Univ. Michigan, Ann Arbor.
14. SCHILLER, J. 1931–37. Dinoflagellatae (Peridineae) in monographis- 37. EVANS, M.S. & D.W. SELL. 1985. Mesh size and collection charac-
cher Behandlung. In L. Rabenhorst, Kryptogamen-Flora. Vol. 10; teristics of 50-cm diameter conical plankton nets. Hydrobiologia
Teil 1 (1931–33); Teil 2 (1935–37). Akademie Verlag, Leipzig, 122:97.
Germany. 38. CLARKE, G.L. & D.F. BUMPUS. 1940. The Plankton Sampler: An
15. SCHILLER, J. 1930. Coccolithineae. In L. Rabenhorst, Kryptogamen- Instrument for Quantitative Plankton Investigations. Spec. Publ. No.
Flora. Vol. 10, p. 89. Akademie Verlag, Leipzig, Germany. 5, Limnological Soc. America.
16. GEITLER, L. 1932. Cyanophyceae von Europa unter Berucksichti- 39. PENNAK, R.W. 1962. Quantitative zooplankton sampling in littoral
gung der anderen Kontinente. In L. Rabenhorst, Kryptogamen- vegetation areas. Limnol. Oceanog. 7:487.
Flora. Vol. 14, p. 1. Akademie Verlag, Leipzig, Germany. 40. HANEY, J.F. & D.J. HALL. 1973. Sugar-coated Daphnia; A preser-
17. WELCH, P.S. 1948. Limnological Methods. Blakiston Co., Philadel- vation technique for Cladocera. Limnol. Oceanogr. 18:331.
phia, Pa. 41. COATS, D.W. & J.F. HEINBOKEL. 1982. A study of reproduction and
18. STRICKLAND, J.D.H. & T.R. PARSONS. 1968. A Practical Manual of other life cycle phenomena in plankton protists using an acridine
Sea Water Analysis. Fish. Res. Board Can. Bull. No. 167. Queen’s orange fluorescence technique. Mar. Biol. 67:71.
Printer, Ottawa, Ont. 42. GANNON, J.E. & S.A. GANNON. 1975. Observations on the narcoti-
19. DUSSART, B.M. 1965. Les différentes catégories de plancton. Hy- zation of crustacean zooplankton. Crustaceana 28(2):220.
drobiologia 26:72. 43. STEEDMAN, H.F. 1976. Narcotizing agents and methods. In H.F.
20. SIEBURTH, J.MCN., V. SMETACEK & J. LENZ. 1978. Pelagic ecosystem Steedman, ed. Zooplankton Fixation and Preservation. Monogr.
structure: Heterotrophic compartments of plankton and their rela- Oceanogr. Methodol. No. 4. United Nations Educational, Scientific
tionship to plankton size fractions. Limnol. Oceanogr. 23: 1256. & Cultural Org., Paris.
10-10 BIOLOGICAL EXAMINATION (10000)

10200 C. Concentration Techniques

The organisms contained in water samples sometimes must be distorted by even gentle filtration. When populations are dense
concentrated in the laboratory before analysis. Three techniques and the content of detritus is high, the filter clogs quickly and silt
for concentrating phytoplankton, namely, sedimentation, mem- may crush the organisms or obscure them from view.
brane filtration, and centrifugation, are described below. A spe- Pour a measured volume of well-mixed sample into a funnel
cial technique for zooplankton also is given. equipped with a membrane filter having a pore diameter of 0.45
␮m. Apply a vacuum of less than 50 kPa to the filter until about
1. Sedimentation 0.5 cm of sample remains on filter. Break vacuum, then apply
low vacuum (about 12 kPa) to remove remaining water but not
Sedimentation is the preferred method of concentration be- to dry the filter.
cause it is nonselective (unlike filtration) and nondestructive For samples with a low phytoplankton and silt content the
(unlike filtration or centrifugation), although many of the pico- method does not require counting of individual plankters to
plankton, the smaller nannoplankton, and actively swimming assemble enumeration data and it increases the probability of
flagellates (in unpreserved samples) may not settle completely. observing less abundant forms.2 Samples also may be concen-
The volume concentrated varies inversely with the abundance of trated on a filter, inverted onto a microscope slide, and quick-
organisms and is related to sample turbidity. It may be as small frozen, permitting the removal of the filter and transfer of plank-
as 1 mL for use with an inverted microscope or as large as 1 L ton to the slide.3,4
for general phytoplankton and zooplankton enumeration.
Allow 1 h settling/mm of column depth. For a treated sample 3. Centrifugation
(10 mL liquid detergent/L) allow about 0.5 h settling/mm depth.1
The sample may be concentrated in a series of steps by quanti- Plankton can be concentrated by batch or continuous centri-
tatively transferring the sediment from the initial container to fugation. Centrifuge batch samples at 1000 g for 20 min. The
sequentially smaller ones. Use cylindrical settling chambers with Foerst continuous centrifuge is no longer recommended as a
thin, clear glass bottoms. Fill settling chambers without forming quantitative device but it may be desirable to continue its use in
a vortex, keep them vibration-free, and move them carefully to existing programs to assure continuity with previously collected
avoid nonrandom distribution of settled matter. Carefully siphon data. Although centrifugation accelerates sedimentation, it may
or decant the supernatants to obtain the desired final volume (5 damage fragile organisms.
mL for diatom mounts). Store the concentrated sample in a
closed, labeled glass vial. 4. Zooplankton Concentration

2. Membrane Filtration Zooplankton samples often need to be concentrated in the field,

especially when large water bottles or pump methods of sampling
The filtration method permits use of high magnification for are used. Moreover, samples obtained by nets or other methods
enumerating small plankters including flagellates and cyanobac- sometimes need to be concentrated further for storage or preparation
teria. However, delicate forms such as “naked” flagellates are for examination. When only small volume reductions are needed,
pour sample back into the bucket of traps or nets. In processing
large volumes of water as with pump sampling, use larger plankton
buckets or funnels with greater water volume retention and filtration
surface area. Construct a filter funnel similar to that shown in Figure
10200:5 of clear acrylic plastic or other suitable material.5 The
volume of the apparatus and the mesh size depend on volume of
water to be filtered and size of organisms to be retained. The mesh
size of the filter funnel normally is the same as that of the net or
other field sampling device.

5. References

1. FURET, J.E. & K. BENSON-EVANS. 1982. An evaluation of the time

required to obtain sedimentation of fixed algal particles prior to
enumeration. Brit. Phycol. J. 17:253.
2. MCNABB, C.D. 1960. Enumeration of freshwater phytoplankton con-
centrated on the membrane filter. Limnol. Oceanogr. 5:57.
3. HEWES, C.D. & O. HOLM-HANSEN. 1983. A method for recovering
nanoplankton from filters for identification with the microscope: The
filter-transfer-freeze (FTF) technique. Limnol. Oceanogr. 28:389.
4. HEWES, C.D., F.M.H. REID & O. HOLM-HANSEN. 1984. The quantitative
Figure 10200:5. Filter funnel for concentrating zooplankton. This device,
analysis of nanoplankton: A study of methods. J. Plankton Res. 6:601.
originally designed for rotifers, can be modified for other
5. LIKENS, G.E. & J.J. GILBERT. 1970. Notes on quantitative sampling of
zooplankters by changing the dimensions and mesh size.
natural populations of planktonic rotifers. Limnol. Oceanogr. 15:816.
(After Likens and Gilbert.5)
PLANKTON (10200)/Preparing Slide Mounts 10-11

10200 D. Preparing Slide Mounts

1. Phytoplankton Semi-Permanent Wet Mounts the residue on the cover glass on a hot plate at 300 to 500°C;
alternatively, use a muffle furnace. This usually requires 20 to 45
Agitate the settled sample concentrate and withdraw a sub- min. Mount as described below.
sample with an accurately calibrated pipet. Clean pipet regularly. Treat samples concentrated for diatom analysis by membrane
To prepare wet mounts transfer 0.1 mL to a glass slide, place a filtration as described by Patrick and Reimer.4 Mix equal vol-
cover slip over the sample, and ring the cover slip with an umes of conc nitric acid (HNO3) and sample. CAUTION: When
adhesive such as clear nail polish to prevent evaporation. For working with conc HNO3 wear safety goggles and an acid-
semipermanent mounts, add a few drops of glycerin to the slide. resistant apron and gloves, and work under a hood. Add a few
As the sample ages the water evaporates, leaving the organisms grains of potassium dichromate (K2Cr2O7)5 to facilitate digestion
imbedded in the glycerin. If the cover slip is ringed with adhe- of the filter and cellular organic matter. Add more dichromate if
sive, the slide can be retained for a few years if stored in the solution color changes from yellow to green. Place sample on a
dark. hot plate and boil down to approximately one-third the original
volume. Alternatively, let treated sample stand overnight. This
cleaning process destroys organic matter and leaves only diatom
2. Phytoplankton Permanent Mounts
shells (frustules). Cool, wash with distilled water, and mount as
described above. Transfer cleaned frustules to a cover glass and
a. Membrane filter mounts: Place two drops of immersion oil dry as described above.
on a labeled slide. Immediately after filtering place the filter on Place a drop of mounting medium in the center of a labeled
top of the oil with a pair of forceps and add two drops of oil on slide. Use 25- by 75-mm slides with frosted ends. Using a
top of the filter. The oil impregnates the filter and makes it suitable high-refractive-index microscopic mounting medium as-
transparent. Impregnation time is 24 to 48 h. This procedure can sures permanent, easily handled mounts for examination under
be completed in 1 to 2 h by applying heat (70°C). Once the filter oil immersion. Heat the slide to near 90°C for 1 to 2 min before
has cleared, place a few additional drops of oil on it and cover applying the heated cover slip with its sample residue to hasten
with a cover slip. The mounted filter is now ready for micro- evaporation of solvent in the mounting medium. Remove the
scopic examination. Alternatively, mount membrane filters in slide to a cool surface and, during cooling (5 to 10 s), apply firm
mounting medium.* Immerse filters in 1-propanol to displace but gentle pressure to the cover glass with a broad, flat instru-
residual water and transfer to xylol for several minutes to clear ment.
filters. Place a section of filter or entire filter on a microscope
slide with the mounting medium, cover with a cover glass, and
dry at low temperature.1 4. Zooplankton Mounts
b. Sedimented slide mounts: Two techniques are available for
making permanent, resin mounts of natural phytoplankton that For zooplankton analyses, withdraw a 5-mL subsample from
has been deposited by sedimentation on a microscope slide or the concentrate and dilute or concentrate further as necessary.
cover glass and dehydrated by ethanol vapor substitution.2,3 Transfer sample to a counting cell or chamber (see below) for
analysis as a wet mount. Use polyvinyl lactyl phenol† for pre-
3. Diatom Mounts paring semipermanent zooplankton mounts. The mounts are
good for about a year, after which time the clearing agent causes
Samples concentrated for diatom analysis by settling or cen- deterioration of organisms. For long-term storage ring cover slip
trifugation may contain dissolved materials, such as marine salts, with clear lacquer (fingernail polish) to retard mountant crystal-
formalin, and detergents, that will leave interfering residues. lization. For permanent mounting, other mountants are avail-
Wash well with distilled water before slide preparation. Transfer able.‡
several drops of washed concentrate by means of a large-bore For the protozoan portion of the microzooplankton, a protargol
disposable pipet or large-bore dropper to a cover glass on a hot staining procedure6 not only provides a permanent mount but
plate warmed enough to increase the evaporation rate but not also reveals the cytological details often necessary for identifi-
enough to cause boiling (use a large-bore pipet or dropper to cation. This procedure is qualitative and is especially important
prevent possible selective filtration, thus exclusion, of larger in taxonomic studies of the ciliated protozoa.
forms or those forming colonies or chains). If the cleaned ma-
terial is very concentrated, improve distribution of diatoms by
adding the drops to a cover glass already flooded with distilled 5. References
water. Evaporate to dryness. Repeat addition and evaporation
until a sufficient quantity of sample has been transferred to the 1. MILLIPORE FILTER CORPORATION. 1966. Biological examination of wa-
cover glass, but avoid producing a residue so dense that organ- ter, sludge and bottom materials. Millipore Techniques, Water Mi-
crobiology, p. 25.
isms cannot be recognized. If in doubt about the density, exam-
ine under a compound microscope. After evaporation, incinerate
† Biomedical Specialists, Box 1687, Santa Monica, CA.
‡ CMC-10, Master’s Chemical Co., P.O. Box 2382, Des Plaines, IL; Hydramount,
* Permount, Fisher Scientific Co., or equivalent. Biomedical Specialists, Box 1687, Santa Monica, CA; or equivalent.
10-12 BIOLOGICAL EXAMINATION (10000)

2. SANFORD, G.R., A. SANDS & C.R. GOLDMAN. 1962. A settle-freeze 5. HOHN, M.H. & J. HELLERMAN. 1963. The taxonomy and structure of
method for concentrating phytoplankton in quantitative studies. Lim- diatom populations for three eastern North American rivers using
nol. Oceanogr. 14:790. three sampling methods. Trans. Amer. Microsc. Soc. 62:250.
3. CRUMPTON, W.G. & R.G. WETZEL. 1981. A method for preparing 6. SMALL, E.B. & D.H. LYNN. 1985. Phylum Ciliophora Doflein, 1901.
permanent mounts of phytoplankton for critical microscopy and cell In J.J. Lee, S.H. Hunter & E.C. Bovee, eds. An Illustrated Guide to
counting. Limnol. Oceanogr. 26:976. the Protozoa. Soc. Protozoology, Lawrence, Kansas.
4. PATRICK, R. & C.W. REIMER. 1967. The Diatoms of the United States.
Vol. 1. Monogr. 13, Philadelphia Acad. Natur. Sci., Philadelphia, Pa.

10200 E. Microscopes and Calibrations

1. Compound Microscope appropriate size depends on the density of organisms. After a

suitable period of settling (see Section 10200C.1), count organ-
Use either a standard or an inverted compound microscope for isms in the settling chamber.
algal identification and enumeration. Equip either type with a The major advantage of the inverted microscope is that by a
mechanical stage capable of moving all parts of a counting cell simple rotation of the nosepiece a specimen can be examined (or
past the objective lens. Standard equipment is a set of 10⫻ or counted) directly in the settling chamber at any desired magni-
12.5⫻ oculars and 10⫻, 20⫻, 40⫻, and 100⫻ objectives. Use fication. Although not recommended, oil immersion objectives
objectives to provide adequate working distance for the counting have some useful applications. No preparation or manipulation
chamber. Magnification requirements vary with the plankton other than settling is required. Generally, examine a preserved
fraction being investigated, the type of microscope, counting sample. Techniques are available for samples with an abundance
chamber used, and optics. With standard objectives, the Sedg- of organisms that tend to float.4
wick-Rafter chamber limits magnification to approximately
200⫻ and the Palmer-Maloney cell limits magnification to ap- 4. Epifluorescence Microscope
proximately 500⫻. Inverted microscopes are limited in resolu-
tion by their optics. The useful upper limit of magnification for An epifluorescence microscope may be either standard or in-
any objective is 1000 times the numerical aperture (NA). Above verted. It uses incident light to excite electrons in intracellular
this magnification, no greater detail can be resolved. Use com- compounds, such as pigments or absorbed stains, with the energy
binations of oculars, intermediate magnifiers, and objectives to emitted during electron return to the ground state being measured as
obtain the greatest magnification without exceeding the useful fluorescent light. The technique has been applied to the microscopic
limit of magnification. When the limit is exceeded, empty mag- identification of chlorophyll-containing cells (autotrophs) and non-
nification results. Empty magnification occurs where the image pigmented heterotrophic plankton; fluorescent stains such as primu-
is larger but no greater resolution is achieved. Optics providing lin or proflavin also have been used to differentiate nannoplanktonic
contrast enhancement such as phase contrast or differential in- primary and secondary producers.5–7 Excitation and emission wave-
terference contrast are useful. lengths are unique for each pigment and stain and require distinct
light filter combinations and light sources. Select the filter combi-
2. Stereoscopic Microscope nations for the particular application. Epifluorescence microscopy is
particularly useful for the enumeration of picoplankton and hetero-
The stereoscopic microscope is essentially two complete mi- trophic flagellate populations common to most aquatic systems.
croscopes assembled into a binocular instrument to give a ste- Concentrate samples by membrane filtration. Use epifluorescence
reoscopic view and an erect rather than an inverted image. Use microscopy as a complementary procedure to standard light micro-
this microscope for the study and counting of large plankters scope counting techniques.
such as mature microcrustacea. Include 10⫻ to 15⫻ paired
oculars in combination with 1⫻ to 8⫻ objectives. This combi- 5. Microscope Calibration
nation of optics bridges the gap between the hand lens and the
compound microscope and provides magnification ranging from Microscope calibration is essential. The usual equipment for
10⫻ to 120⫻. Alternatively, use a good-quality zoom-type in- calibration is a Whipple grid (ocular micrometer, reticle, or reticule)
strument with comparable magnification. placed in an eyepiece of the microscope and a stage micrometer that
has a standardized, accurately ruled scale on a glass slide. The
3. Inverted Compound Microscope Whipple disk (Figure 10200:6) has an accurately ruled grid subdi-
vided into 100 squares. One square near the center is subdivided
The inverted compound microscope often is used routinely for further into 25 smaller squares. The outer dimensions of the grid are
plankton counting in many laboratories.1–3 This instrument is such that with a 10⫻ objective and a 10⫻ ocular, it delimits an area
unique in that the objectives are below a movable stage and the of approximately 1 mm2 on the microscope stage. Because this area
illumination comes from above, thus permitting viewing of or- may differ from one microscope to another, carefully calibrate the
ganisms that have settled to the bottom of a chamber. Place Whipple grid for each microscope.
samples in a cylindrical settling chamber having a thin, clear With the ocular and stage micrometers parallel and in part
glass bottom. Chambers of various capacities are available; the superimposed, match the line at the left edge of the Whipple grid
PLANKTON (10200)/Phytoplankton Counting Techniques 10-13

Figure 10200:6. Ocular micrometer ruling. A Whipple micrometer reti-

cule is illustrated.

with the zero mark on the stage micrometer scale (Figure 10200:
7). Determine the width of the Whipple grid image to the nearest
0.01 mm from the stage micrometer scale. Should the width of Figure 10200:7. Calibration of Whipple Square, as seen with 10⫻ ocular and
43⫻ objective (approximately 430⫻ total magnification).
the image of the Whipple grid be exactly 1 mm (1000 ␮m), the
larger squares will be 1/10 mm (100 ␮m) on a side and each of
the smaller squares 1/50 mm (20 ␮m). ples of picoplankton from the Laurentian Great Lakes. J. Great Lakes
When the microscope is calibrated at higher magnifications, Res. 10:90.
4. REYNOLDS, C.S. & G.H.M. JAWORSKI. 1978. Enumeration of natural
the entire scale on the stage micrometer will not be seen; make
Microcystis populations. Brit. Phycol. J. 13:269.
measurements to the nearest 0.001 mm. Additional details for 5. DAVIS, P.G. & J. McN. SIEBURTH. 1982. Differentiation of phototro-
calibration are available.8 phic and heterotrophic nanoplankton populations in marine waters by
epifluorescence microscopy. Ann. Inst. Oceanogr. 58:249.
6. References 6. CARON, D.A. 1983. Techniques for enumeration of heterotrophic and
phototrophic nanoplankton, using epifluorescence microscopy, and
1. WETZEL, R.G. & G.E. LIKENS. 1991. Limnological Analyses, 2nd ed. comparison with other procedures. Appl. Environ. Microbiol. 46:491.
Springer-Verlag, New York, N.Y. 7. SHERR, E.B. & B.F. SHERR. 1983. Double-staining epifluorescence
2. LUND, J.W.G., C. KIPLING & E.D. LECREN. 1958. The inverted mi- techniques to assess frequency of dividing cells and bacteriovory in
croscope method of estimating algal numbers and the statistical basis natural populations of heterotrophic microprotozoa. Appl. Environ.
of estimations by counting. Hydrobiologia 11:143. Microbiol. 46:1388.
3. SICKO-GOAD, L. & E.F. STOERMER. 1984. The need for uniform 8. JACKSON, H.W. & L.G. WILLIAMS. 1962. Calibration and use of certain
terminology concerning phytoplankton cell size fractions and exam- plankton counting equipment. Trans. Amer. Microsc. Soc. 81:96.

10200 F. Phytoplankton Counting Techniques

1. Counting Units
Enumeration Reporting
Some phytoplankton are unicellular while others are mul- Method Counting Unit Unit
ticellular (colonial). The variety of configurations poses a
Total cell count One cell Cells/mL
problem in enumeration. For example, should a four-celled Natural unit count1 One organism (any Units/mL
colony of Scenedesmus (Plates 32, 34) be reported as one (clump count) unicellular organism
colony or four individual cells? Listed below are suggestions or natural colony)
for reporting: Areal standard unit 400 ␮m2 Units/mL
count*
*Areal standard unit equals area of four small squares in Whipple grid at a
magnification of 200.
10-14 BIOLOGICAL EXAMINATION (10000)

Making a total cell count is time-consuming and tedious,

especially when colonies consist of thousands of individual cells.
The natural unit or clump is the most easily used system;
however, it is not necessarily accurate because sample handling
and preserving may dislodge cells from the colony. The unit
method also may not be quantitatively accurate nor reflect abun-
dance of biomass or biovolume. Whatever method is chosen,
identify it in reporting results.
If the distribution of organisms is random and the population
fits a Poisson distribution, the counting error may be estimated.2
For example, the approximate 95% confidence limits, as a per-
centage of the number of units counted (N), equals:

2
(100%)
冑N
Figure 10200:8. Counting cell (Sedgwick-Rafter), showing method of
filling. Source: WHIPPLE, G.C., G.M. FAIR & M.C.
WHIPPLE. 1927. The Microscopy of Drinking Water. John
Thus, if 100 units are counted, the 95% confidence limits
Wiley & Sons, New York, N.Y.
approximate ⫾ 20%. For a count of 400 units, the limits are
about 10%.

2. Counting Procedures sample with a large-bore pipet (Figure 10200:8). Placing cover
slip in this manner will help prevent formation of air bubbles in
To enumerate plankton use a counting cell or chamber that cell corners. The cover slip often will rotate slowly and cover the
limits the volume and area for ready calculation of population inner portion of the S-R cell during filling. Do not overfill
densities. because this would yield a depth greater than 1 mm and produce
When counting with a Whipple grid, establish a convention for an invalid count. Do not permit large air spaces caused by
tallying organisms lying on an outer boundary line. For example, in evaporation to develop in the chamber during a lengthy exami-
counting a “field” (entire Whipple square), designate the top and left nation. To prevent formation of air spaces, occasionally place a
boundaries as “no-count” sides, and the bottom and right boundaries small drop of distilled water on edge of cover glass.
as “count” sides. Thus, tally every plankter touching a “count” side Before counting let the S-R cell stand for at least 15 min to
from the inside or outside but ignore any touching a “no-count” settle plankton. Count plankton on the bottom of the S-R cell.
side. If significant numbers of filamentous or other large forms cross Some phytoplankton, notably some blue-green algae or motile
two or more boundaries of the grid, count them separately at a lower flagellates in unpreserved samples, may not settle but rise to the
magnification and include their number in the total count. underside of the cover slip. When this occurs, count these
To identify organisms use standard bench references (see organisms and add to total of those counted on the cell bottom to
Section 10900). derive total number of organisms. Count algae in strips or fields.
Do not count dead cells or broken diatom frustules. Tally 2) Strip counting—A “strip” the length of the cell constitutes
empty centric and pennate diatoms separately as “dead centric a volume approximately 50 mm long, 1 mm deep, and the width
diatoms” or “dead pennate diatoms” for use in converting the of the total Whipple grid.
diatom species proportional count to a count per milliliter. The number of strips to be counted is a function of the
Magnification is important in phytoplankton identification and precision desired and the number of units (cells, colonies, or
enumeration. Although magnifications of 100⫻ to 200⫻ are useful filaments) per strip. Derive number of plankton in the S-R cell
for counting large organisms or colonies, much higher magnifica- from the following:
tions often are required. It is useful to categorize techniques for
phytoplankton counting according to the magnifications provided. C ⫻ 1000 mm3
a. Low-magnification (up to 200⫻) methods: The Sedgwick- No./mL ⫽
L⫻D⫻W⫻S
Rafter (S-R) cell is a device commonly used for plankton counting
because it is easily manipulated and provides reasonably reproduc-
ible data when used with a calibrated microscope equipped with an where:
C ⫽ number of organisms counted,
eyepiece measuring device such as the Whipple grid.
L ⫽ length of each strip (S-R cell length), mm,
The greatest disadvantage associated with the cell is that
D ⫽ depth of a strip (S-R cell depth), mm,
objectives providing high magnification cannot be used. As a
W ⫽ width of a strip (Whipple grid image width), mm, and
result, the S-R cell is not appropriate for examining nannoplank- S ⫽ number of strips counted.
ton. The S-R cell is approximately 50 mm long by 20 mm wide
by 1 mm deep. The total area of the bottom is approximately Multiply or divide number of cells per milliliter by a correc-
1000 mm2 and the total volume is approximately 1000 mm3 or tion factor to adjust for sample dilution or concentration.
1 mL. Carefully check the exact length and depth of the cell with 3) Field counting—On samples containing many plankton (10
a micrometer and calipers before use. or more plankters per field), make field counts rather than strip
1) Filling the cell—Before filling the S-R cell with sample, counts. Count plankters in random fields each consisting of one
place the cover glass diagonally across the cell and transfer Whipple grid. The number of fields counted will depend on
PLANKTON (10200)/Phytoplankton Counting Techniques 10-15

plankton density and statistical accuracy desired (see 10200F.1). scope chambers, membrane filter mounts, sedimented slide
Calculate the number of plankton per milliliter as follows: mounts, the Lackey drop method, and diatom mounts.
1) Inverted microscope counts—Prepare a sample for exami-
nation by filling the settling chamber. After the desired settling
C ⫻ 1000 mm3
No./mL ⫽ time (see Section 10200C.1), transfer the chamber to the micro-
A⫻D⫻F scope stage. Count perpendicular strips across the center of the
bottom cover glass. Strip counts may be made by using a
where: Whipple grid or special counting oculars that have a pair of
C ⫽ number of organisms counted, adjustable parallel hairs and a single cross hair. Determine the
A ⫽ area of a field (Whipple grid image area), mm2, width of the strip with a stage micrometer and tally organisms as
D ⫽ depth of a field (S-R cell depth), mm, and they pass the single cross hair that functions as a reference point.
F ⫽ number of fields counted. Hold strip width constant for any series of samples. Alternatively
examine random nonoverlapping fields until at least 100 units of
Multiply or divide the number of cells per milliliter by a
the dominant species are counted. For highest accuracy, partic-
correction factor to adjust for sample dilution or concentration.
ularly because algae distribution may be nonuniform, count the
b. Intermediate magnification (low to 500⫻) methods: The
entire chamber floor. Alternatively, make a random field-
Palmer-Maloney (P-M) nannoplankton cell3 is designed specif-
minimum count to attain a precision level of at least 85%.4
ically for nannoplankton enumeration. It has a circular chamber
with a 17.9-mm diam, 0.4-mm depth, and 0.1-mL volume. The
C ⫻ At
shallow depth permits use of 40 to 45⫻ objectives with sufficient Strip count (No./mL) ⫽
working distance. The principal disadvantage of the P-M cell is L⫻W⫻S⫻V
that these magnifications (400 to 450⫻) often are insufficient for
nannoplankton identification and enumeration. where:
Because a relatively small sample portion is examined in the P-M C ⫽ number of organisms counted,
cell do not use it unless the sample contains a dense population (10 At ⫽ total area of bottom of settling chamber, mm2,
L ⫽ length of a strip, mm,
or more plankters per field). Such a small sample portion from a less
W ⫽ width of a strip (Whipple grid image width), mm,
dense population causes serious underestimation of density.
S ⫽ number of strips counted, and
Introduce sample with a pipet into one of the 2- by 5-mm
V ⫽ volume of sample settled, mL.
channels on the side of the chamber with the cover slip in place.
After a 10-min settling period count the plankters in random
fields, with the number of fields depending on density and C ⫻ At
variety of plankton and the statistical accuracy desired. Strips Field count (No./mL) ⫽
Af ⫻ F ⫻ V
may be counted in this or any other circular cell by measuring
the effective diameter and counting two perpendicular strips that where:
cross at the center. Calculate the number per milliliter as follows: Af ⫽ area of a field (Whipple grid image area), mm2,
F ⫽ number of fields counted,
C ⫻ 1000 mm3 and other terms are as defined above.
No./mL ⫽
A⫻D⫻F 2) Membrane filter mounts—Concentrate sample as directed
in Section 10200C.2 and prepare membrane filter as directed in
where: Section 10200D.2a.
C ⫽ number of organisms counted, Examine samples, concentrated on unlined membrane filters
A ⫽ area of a field (Whipple grid image), mm2, and mounted in oil as described above. Count enough random
D ⫽ depth of a field (P-M cell depth), mm, and fields to ensure desired level of statistical accuracy (see
F ⫽ number of fields counted. 10200F.1). Select magnification level and size of microscope
field (quadrat) such that the most abundant species appear in at
Multiply or divide the number of cells per milliliter by a least 70% but not more than 90% of microscopic fields examined
correction factor to adjust for sample dilution or concentration. (80% is optimum). Adjust microscope field size by using part or
Another readily available chamber is the standard medical hema- all of the Whipple grid. Examine 30 random microscope fields
cytometer used for enumerating blood cells. It has a ruled grid and record number of fields in which each species occurred.
machined into a counting plate and is fitted with a ground-glass Report results as organisms per milliliter, calculated as follows:
cover slip. The grid is divided into 1-mm2 divisions; the chamber is
0.1 mm deep. Introduce sample by pipet and view under 450⫻
N⫻Q
magnification. Count all cells within the grid. The chamber comes No./mL ⫽
V⫻D
from the manufacturer with a detailed instruction sheet containing
directions on calculations and proper usage. A disadvantage to these
counting cells is that the sample must have a very high plankton where:
N ⫽ density (organisms/field) from Table 10200:II,
density to yield statistically reliable data.
Q ⫽ number of fields per filter,
c. High-magnification methods: Examination of phytoplank-
V ⫽ milliliters filtered, and
ton at high magnification requires the use of oil immersion D ⫽ dilution factor (0.96 for 4% formalin preservative).
objectives. Suitable procedures include using inverted micro-
10-16 BIOLOGICAL EXAMINATION (10000)

TABLE 10200:II. CONVERSION TABLE FOR MEMBRANE FILTER TECHNIQUE 5) Diatom mounts—Prepare samples as directed in Section
(Based on 30 Scored Fields) 10200D.3.
Total F* For diatom species proportional count, examine diatom sam-
Occurrence % N† ples under oil immersion at a magnification of at least 900⫻.
Scan lateral strips the width of the Whipple grid until at least 250
1 3.3 0.03 cells are counted. Available time and accuracy required dictate
2 6.7 0.07 the number of cells to be counted. Determine percentage abun-
3 10.0 0.10 dance of each species from tallied counts and calculate counts
4 13.3 0.14
per milliliter of each species by multiplying percent abundance
5 16.7 0.18
6 20.0 0.22 by total live and dead diatom count obtained from the plankton
7 23.3 0.26 counting chamber. For greater accuracy distinguish between
8 26.7 0.31 living and dead diatoms at the species level.
9 30.0 0.35 6) Phytoplankton staining technique—Staining algae permits
10 33.3 0.40 differentiation between “live” and “dead” diatoms.6 This permits
11 36.7 0.45 enumerating total phytoplankton in a single sample without
12 40.0 0.51 sacrificing detailed diatom taxonomy. It also results in perma-
13 43.3 0.57 nent reference slides. The procedure is most useful when diatoms
14 46.7 0.63 are major components of phytoplankton and it is important to
15 50.0 0.69
distinguish between living and dead diatoms.
16 53.3 0.76
17 56.7 0.83 Preferably preserve samples in Lugol’s solution or alternatively
18 60.0 0.91 in formalin (see 10200B.3). For analysis thoroughly mix the sample
19 63.3 1.00 and filter a portion through a 47-mm-diam membrane filter (pore
20 66.7 1.10 diam 0.45 or 0.65 ␮m). Use a vacuum of 16 to 20 kPa and never let
21 70.0 1.20 sample dry. Add 2 to 5 mL aqueous acid fuchsin solution (dissolve
22 73.3 1.32 1 g acid fuchsin in 100 mL distilled water to which 2 mL glacial
23 76.7 1.47 acetic acid has been added; filter) to the filter and let stand for 20
24 80.0 1.61 min. After staining, filter sample, wash briefly with distilled water,
25 83.3 1.79 and filter again. Administer successive rinses of 50%, 90%, and
26 86.7 2.02
100% propanol to the sample while filtering. Soak for 2 min in a
27 90.0 2.30
28 93.3 2.71 second 100% propanol wash, filter, and add xylene. At least two
29 96.7 3.42 washes are required; let the final one soak 10 min before filtering.
30 100.0 ? Trim the xylene-soaked filter and place on a microscope slide on
which there are several drops of mounting medium.† Apply several
total number of species occurrences ⫻ 100
*F⫽ more drops of medium to top of filter and install a cover glass.
total number of fields examined
† N ⫽ number of organisms per field.
Carefully squeeze out excess mounting medium. Make the final
mount permanent by lacquering the edges of the cover glass.
Count organisms using the most appropriate magnification.
“Live” diatoms typically are red while “dead” ones are un-
3) Sedimented slide mounts—Examine mounts prepared as stained. Oil immersion is necessary for species identifications of
directed in Section 10200D.2b. diatoms and many other algae. Count either strips or random
4) Lackey drop method—The Lackey drop (microtransect) fields and calculate plankton densities per milliliter:
method5 is a simple method of obtaining counts of considerable
accuracy with samples containing a dense plankton population. It C ⫻ At
No./mL ⫽
is similar to the S-R strip count. Ac ⫻ V
Prepare slides as directed in Section 10200D.1. Oil immersion
objectives can be used with the semipermanent slides. Count where:
organisms in enough strips to ensure desired level of statistical C ⫽ number of organisms counted,
At ⫽ total area of effective filter before trimming and mounting,
accuracy (see 10200F.1). Calculate number of organisms per
Ac ⫽ area counted (strips or fields), and
milliliter as follows:
V ⫽ volume of sample filtered, mL.

C ⫻ At 3. References
No./mL ⫽
As ⫻ S ⫻ V 1. INGRAM, W.M. & C.M. PALMER. 1952. Simplified procedures for
collecting, examining, and recording plankton in water. J. Amer.
where: Water Works Assoc. 44:617.
2. STRICKLAND, J.D.H. & T.R. PARSONS. 1968. A Practical Manual of Sea
C ⫽ number of organisms counted,
Water Analysis. Fish. Res. Board Can. Bull. No. 167. Queen’s
At ⫽ area of cover slip, mm2,
Printer, Ottawa, Ont.
As ⫽ area of one strip, mm2,
S ⫽ number of strips counted, and
V ⫽ volume of sample under the cover slip, mL.
† Permount, Fisher Scientific Co., or equivalent.
PLANKTON (10200)/Zooplankton Counting Techniques 10-17

3. PALMER, C.M. & T.E. MALONEY. 1954. A New Counting Slide for Nanno- 5. LACKEY, J.B. 1938. The manipulation and counting of river plankton
plankton. Spec. Publ. No. 21, American Soc. Limnology & Oceanography. and changes in some organisms due to formalin preservation. Pub.
4. SOURNIA, A., ed. 1978. Phytoplankton Manual. Monogr. Oceanogr. Meth- Health Rep. 53:2080.
odol. No. 6. United Nations Educational, Scientific & Cultural Org., Paris. 6. OWEN, B.B., JR., M. AFZAL & W.R. CODY. 1978. Staining prepara-
tions for phytoplankton and periphyton. Brit. Phycol. J. 13:155.

10200 G. Zooplankton Counting Techniques

1. Subsampling a 1- to 5-mL clear acrylic plastic counting cell fitted with a glass
cover slip. For larger, mature microcrustacea use a counting
Count entire samples having low zooplankton numbers (⬍200 chamber holding 5 to 10 mL. A Sedgwick-Rafter cell is not
zooplankters) without subsampling. However, most zooplankton suitable because of size. An open counting chamber 80 by 50
samples will contain more organisms than can be enumerated
practically; therefore, use a subsampling procedure. Before sub-
sampling, remove and enumerate all large uncommon organisms
such as fish larvae in fresh water or coelenterates, decapods, fish
larvae, etc., in salt water. Subsample by the pipet or splitting
method.
In the pipet method, adjust sample to a convenient volume in
a graduated cylinder or Imhoff cone. Concentrating the plankton
by using a rubber bulb and clear acrylic plastic tube with fine
mesh netting fitted on the end is convenient and accurate (Figure
10200:9). For picoplankton and the smaller microzooplankton,
use sedimentation techniques described for concentrating phyto-
plankton. Transfer sample to a beaker or other wide-mouth
vessel for subsampling with a Hensen-Stempel or similar wide-
bore pipet. Gently stir sample completely and randomly with the
pipet and quickly withdraw 1 to 5 mL. Transfer to a suitable
counting chamber.
Alternatively, subsample by splitting with any of a number of
devices of which the Folsom plankton splitter1 is best known
(Figure 10200:10). Level splitter before using. Place sample in
the splitter and divide into subsplits. Rinse splitter into the
subsamples. Repeat until a workable number (200 to 500 indi-
viduals) is obtained in a subsample. Exercise care to provide
unbiased splits. Even when using the Folsom splitter unbiased
subsamples cannot be unquestioningly assumed;2 therefore,
count animals in several subsamples from the same sample to
verify that the splitter is unbiased and to determine the sampling
error introduced by using it.
Another method permits abundance estimates of more equiv-
alent levels of precision among taxa than obtained with either the
Hensen-Stempel pipet or the Folsom splitter.3 Normal counting
procedures tally organisms on the basis of their abundance in a
sample. Therefore, in a sample with a dominant organism mak-
ing up 50% of total numbers, the tally of the dominant taxon will
be large and have a small error. However, error about the
subdominants will increase as the tally of each taxon decreases.
By accepting one level of precision, the technique3 has been
developed to obtain the same error about dominants and sub-
dominants, permitting quantitative comparisons between taxa
over successive times or between stations.
Figure 10200:9. A simple, efficient device for concentrating plankton. The tube
is lowered into the beaker containing the sample. Water filtering
2. Enumeration into the tube is removed with the rubber bulb. The filter is nylon
monofilament screen cloth that is glued to the bottom of the tube.
Using a compound microscope and a magnification of 100⫻, The mesh size should be sufficiently small to prevent zooplank-
enumerate small zooplankton (protozoa, rotifers, and nauplii) in ters from entering the filtrate (after Dodson and Thomas5).
10-18 BIOLOGICAL EXAMINATION (10000)

Figure 10200:10. The Folsom plankton splitter.

mm and 2 mm deep is desirable; however, an open chamber is V⬙ ⫽ volume counted, mL, and
difficult to move without jarring and disrupting the count. A mild V⵮ ⫽ volume of the grab sample, m3.
detergent solution placed on the chamber before counting re-
To obtain organisms per liter divide by 1000.
duces organism movements or special counting trays with par-
allel or circular grooves or partitions4,5 can be used. Count
microcrustacea with a binocular dissecting microscope at 20⫻ to 3. References
40⫻ magnification. If identification is questionable, remove or-
ganisms with a microbiological transfer loop and examine at a 1. LONGHURST, A.R. & D.L.R. SEIBERT. 1967. Skill in the use of Fol-
higher magnification under a compound microscope. som’s plankton sample splitter. Limnol. Oceanogr. 12:334.
Report smaller zooplankton as number per liter and larger 2. MCEWEN, G.F., M.W. JOHNSON & T.R. FOLSOM. 1954. A statistical
forms as number per cubic meter: analysis of the Folsom sample splitter based upon test observations.
Arch. Meteorol. Geophys. Bioklimatol., Ser. A, 6:502.
C ⫻ V⬘ 3. ALDEN, R.W., III, R.C. DAHIYA & R.J. YOUNG, JR. 1982. A method for
No./m3 ⫽ the enumeration of zooplankton samples. J. Exp. Mar. Biol. Ecol.
V⬙ ⫻ V⵮ 59:185.
4. GANNON, J.E. 1971. Two counting cells for the enumeration of
where: zooplankton micro-crustacea. Trans. Amer. Microsc. Soc. 90:486.
C ⫽ number of organisms counted, 5. DODSON, A.N. & W.H. THOMAS. 1964. Concentrating plankton in
V⬘ ⫽ volume of the concentrated sample, mL, gentle fashion. Limnol. Oceanogr. 9:455.

10200 H. Chlorophyll

The concentration of photosynthetic pigments is used exten- in phytoplankton include chlorophylls b and c, xanthophylls,
sively to estimate phytoplankton biomass.1,2 All green plants phycobilins, and carotenes. The important chlorophyll degrada-
contain chlorophyll a, which constitutes approximately 1 to 2% tion products found in the aquatic environment are the chloro-
of the dry weight of planktonic algae. Other pigments that occur phyllides, pheophorbides, and pheophytins. The presence or
PLANKTON (10200)/Chlorophyll 10-19

absence of the various photosynthetic pigments is used, among 4) Filtration equipment, filters, glass fiber† or membrane
other features, to separate the major algal groups. (0.45-␮m porosity, 47-mm diam); vacuum pump; solvent-resis-
The three methods for determining chlorophyll a in phyto- tant disposable filter assembly, 1.0-␮m pore size;‡ 10-mL sol-
plankton are the spectrophotometric,3–5 the fluorometric,6 – 8 and vent-resistant syringe.
the high-performance liquid chromatographic (HPLC) tech- 5) Saturated magnesium carbonate solution: Add 1.0 g finely
niques.9 Fluorometry is more sensitive than spectrophotometry, powdered MgCO3 to 100 mL distilled water.
requires less sample, and can be used for in-vivo measure- 6) Aqueous acetone solution: Mix 90 parts acetone (reagent-
ments.10 These optical methods can significantly under- or over- grade BP 56°C) with 10 parts saturated magnesium carbonate
estimate chlorophyll a concentrations,11–18 in part because of the solution. For HPLC pigment analysis, mix 90 parts HPLC-grade
overlap of the absorption and fluorescence bands of co-occurring acetone with 10 parts distilled water.
accessory pigments and chlorophyll degradation products. b. Extraction procedure:
Pheophorbide a and pheophytin a, two common degradation 1) Concentrate sample by centrifuging or filtering as soon as
products of chlorophyll a, can interfere with the determination of possible after collection. If processing must be delayed, hold
chlorophyll a because they absorb light and fluoresce in the same samples on ice or at 4°C and protect from exposure to light. Use
region of the spectrum as does chlorophyll a. If these pheopig- opaque bottles because even brief exposure to light during stor-
ments are present, significant errors in chlorophyll a values will age will alter chlorophyll values. Samples on filters taken from
result. Pheopigments can be measured either by spectrophotom- water having pH 7 or higher may be placed in airtight plastic
etry or fluorometry, but in marine and freshwater environments bags and stored frozen for 28 d. Process samples from acidic
the fluorometric method is unreliable when chlorophyll b co- water promptly after filtration to prevent possible chlorophyll
occurs. Upon acidification of chlorophyll b, the resulting fluo- degradation from residual acidic water on filter. Use glassware
rescence emission of pheophytin b is coincident with that of and cuvettes that are clean and acid-free.
pheophytin a, thus producing underestimation and overestima- 2) Place sample in a tissue grinder, cover with 2 to 3 mL 90%
tion of chlorophyll a and pheopigments, respectively. aqueous acetone solution, and macerate at 500 rpm for 1 min.
HPLC is a useful method for quantifying photosynthetic pig- Use TFE/glass grinder for a glass-fiber filter and glass/glass
ments9,13,15,16,19 –21 including chlorophyll a, accessory pigments grinder for a membrane filter.
(e.g., chlorophylls b and c), and chlorophyll degradation prod- 3) Transfer sample to a screw-cap centrifuge tube, rinse
ucts (chlorophyllides, pheophorbides, and pheophytins). Pig- grinder with a few milliliters 90% aqueous acetone, and add the
ment distribution is useful for quantitative assessment of phyto- rinse to the extraction slurry. Adjust total volume to 10 mL, with
plankton community composition and zooplankton grazing ac- 90% aqueous acetone. Use solvent sparingly and avoid excessive
tivity.22 dilution of pigments. Steep samples at least 2 h at 4°C in the
dark. Glass fiber filters of 25- and 47-mm diam§ have dry
1. Pigment Extraction displacement volumes of 0.03 and 0.10 mL, respectively, and
introduce errors of about 0.3 and 1.0% if a 10-mL extraction
Chlorophyll can be extracted with several different solvents, volume is used.
including acetone, ethanol, and methanol. The procedure de- 4) Clarify by filtering through a solvent-resistant disposable
scribed here uses acetone. Conduct this procedure with chloro- filter (to minimize retention of extract in filter and filter holder,
phyll extracts in subdued light to avoid degradation. Use opaque force 1 to 2 mL air through the filter after the extract), or by
containers or wrap with aluminum foil. The pigments are ex- centrifuging in closed tubes for 20 min at 500 g. Decant clarified
tracted from the plankton concentrate with aqueous acetone and extract into a clean, calibrated, 15-mL, screw-cap centrifuge tube
the optical density (absorbance) of the extract is determined with and measure total volume. Proceed as in 2, 3, 4, or 5 below.
a spectrophotometer. The ease with which the chlorophylls are
removed from the cells varies considerably with different algae. 2. Spectrophotometric Determination of Chlorophyll
To achieve consistent complete extraction of the pigments, dis-
rupt the cells mechanically with a tissue grinder. a. Equipment and reagents:
Glass fiber filters are preferred for removing algae from water. 1) Spectrophotometer, with a narrow band (pass) width (0.5 to
The glass fibers assist in breaking the cells during grinding, 2.0 nm) because the chlorophyll absorption peak is relatively
larger volumes of water can be filtered, and no precipitate forms narrow. At a spectral band width of 20 nm the chlorophyll a
after acidification. Inert membrane filters such as polyester filters concentration may be underestimated by as much as 40%.
may be used where these factors are irrelevant. 2) Cuvettes, with 1-, 4-, and 10-cm path lengths.
a. Equipment and reagents: 3) Pipets, 0.1- and 5.0-mL.
1) Tissue grinder:* Successfully macerating glass fiber filters 4) Hydrochloric acid, HCl, 0.1N.
in tissue grinders with grinding tube and pestle of conical design b. Determination of chlorophyll a in the presence of pheophy-
may be difficult. Preferably use round-bottom grinding tubes tin a: Chlorophyll a may be overestimated by including pheopig-
with a matching pestle having grooves in the TFE tip. ments that absorb near the same wavelength as chlorophyll a.
2) Clinical centrifuge. Addition of acid to chlorophyll a results in loss of the magne-
3) Centrifuge tubes, 15-mL graduated, screw-cap.

† Whatman GF/F (0.7 ␮m), GFB (1.0 ␮m), Gelman AE (1 ␮m),23 or equivalent.
* Kontes Glass Co., Vineland, NJ 08360: Glass/glass grinder, Model No. 8855: ‡ Gelman Acrodisc or equivalent.
Glass/TEE grinder, Model 886000; or equivalent. § GF/F or equivalent.
10-20 BIOLOGICAL EXAMINATION (10000)

sium atom, converting it to pheophytin a. Acidify carefully to a 1.7

final molarity of not more than 3 ⫻ 10⫺3M to prevent certain ⫽ ⫽ 2.43
1.7⫺1.0
accessory pigments from changing to absorb at the same wave-
length as pheophytin a.13 When a solution of pure chlorophyll a
is converted to pheophytin a by acidification, the absorption- c. Determination of chlorophyll a, b, and c (trichromatic
peak-ratio (OD664/OD665) of 1.70 is used in correcting the method): Spectrophotometric procedure—Transfer extract to a
apparent chlorophyll a concentration for pheophytin a. 1-cm cuvette and measure optical density (OD) at 750, 664, 647,
Samples with an OD664 before/OD665 after acidification and 630 nm. Choose a cell path length or dilution to give OD664
ratio (664b/665a) of 1.70 are considered to contain no pheo- between 0.1 and 1.0.
phytin a and to be in excellent physiological condition. So- Use the optical density readings at 664, 647, and 630 nm to
lutions of pure pheophytin show no reduction in OD665 upon determine chlorophyll a, b, and c, respectively. The OD reading
acidification and have a 664b/665a ratio of 1.0. Thus, mixtures at 750 nm is a correction for turbidity. Subtract this reading from
of chlorophyll a and pheophytin a have absorption peak ratios each of the pigment OD values of the other wavelengths before
ranging between 1.0 and 1.7. These ratios are based on the use using them in the equations below. Because the OD of the extract
of 90% acetone as solvent. Using 100% acetone as solvent at 750 nm is very sensitive to changes in the acetone-to-water
results in a chlorophyll a before-to-after acidification ratio of proportions, adhere closely to the 90 parts acetone:10 parts water
about 2.0.3 (v/v) formula for pigment extraction. Turbidity can be removed
Spectrophotometric procedure—Transfer 3 mL clarified ex- easily by filtration through a disposable, solvent-resistant filter
tract to a 1-cm cuvette and read optical density (OD) at 750 and attached to a syringe or by centrifuging for 20 min at 500 g.
664 nm. Acidify extract in the cuvette with 0.1 mL 0.1N HCl. Calculate the concentrations of chlorophyll a, b, and c in the
Gently agitate the acidified extract and read OD at 750 and at extract by inserting the corrected optical densities in following
665 nm, 90 s after acidification. The volumes of extract and acid equations:5
and the time after acidification are critical for accurate, consis-
tent results. a) Ca ⫽ 11.85(OD664) ⫺ 1.54(OD647) ⫺ 0.08(OD630)
The OD664 before acidification should be between 0.1 and
1.0. For very dilute extracts use cuvettes having a longer path b) Cb ⫽ 21.03(OD647) ⫺ 5.43(OD664) ⫺ 2.66(OD630)
length. If a larger cell is used, add a proportionately larger
volume of acid. Correct OD obtained with larger cuvettes to 1 c) Cc ⫽ 24.52(OD630) ⫺ 7.60(OD647) ⫺ 1.67(OD664)
cm before making calculations.
Subtract the 750-nm OD value from the readings before (OD
where:
664 nm) and after acidification (OD 665 nm). Ca, Cb, and Cc ⫽ concentrations of chlorophyll a, b, and c,
Using the corrected values calculate chlorophyll a and pheo- respectively, mg/L, and
phytin a per cubic meter as follows: OD664, OD647,
and OD630 ⫽ corrected optical densities (with a 1-cm light
26.7 (664b ⫺ 665a) ⫻ V 1 path) at the respective wavelengths.
Chlorophyll a, mg/m3 ⫽
V2 ⫻ L
After determining the concentration of pigment in the extract,
26.7 [1.7 (665a) ⫺ 664b] ⫻ V 1 calculate the amount of pigment per unit volume as follows:
Pheophytin a, mg/m3 ⫽
V2 ⫻ L
C a ⫻ extract volume, L
where: Chlorophyll a, mg/m3 ⫽
V1 ⫽ volume of extract, L, volume of sample, m3
V2 ⫽ volume of sample, m3,
L ⫽ light path length or width of cuvette, cm, and
664b, 665a ⫽ optical densities of 90% acetone extract before 3. Fluorometric Determination of Chlorophyll a
and after acidification, respectively.
The fluorometric method for chlorophyll a is more sensitive
The value 26.7 is the absorbance correction and equals A ⫻ K than the spectrophotometric method and thus smaller samples
can be used. Calibrate the fluorometer spectrophotometrically
where: with a sample from the same source to achieve acceptable
A ⫽ absorbance coefficient for chlorophyll a at 664 nm ⫽ 11.0, results. Optimum sensitivity for chlorophyll a extract measure-
and ments is obtained at an excitation wavelength of 430 nm and an
K ⫽ ratio expressing correction for acidification.
emission wavelength of 663 nm. A method for continuous mea-
surement of chlorophyll a in vivo is available, but is reported to

冉 冊
664b pure chlorophyll a
665a
be less efficient than the in-vitro method given here, yielding
about one-tenth as much fluorescence per unit weight as the
same amount in solution. Pheophytin a also can be determined
冉 冊 冉 冊
⫽
664b
pure chlorophyll a ⫺
664b
pure pheophytin a
fluorometrically.24
665a 665a a. Equipment and reagents: In addition to those listed under
1a and 2a above:
PLANKTON (10200)/Chlorophyll 10-21

Fluorometer,㛳 equipped with a high-intensity F4T.5 blue Rb ⫽ fluorescence of extract before acidification,
lamp, photomultiplier tube R-446 (red-sensitive), sliding win- Ra ⫽ fluorescence of extract after acidification,
dow orifices 1⫻, 3⫻, 10⫻, and 30⫻, and filters for light emis- r ⫽ Rb/Ra, as determined with pure chlorophyll a for the
sion (CS2-64) and excitation (CS-5-60). A high-sensitivity door instrument (redetermine r and Fs if filters or light source
is preferable. are changed),
b. Extraction procedure: Prepare sample as directed in 1b Ve ⫽ volume of extract, and
Vs ⫽ volume of sample.
above.
1) Calibrate fluorometer with a chlorophyll solution of known d. Extraction of whole water, nonfiltered samples: Alterna-
concentration as follows: Prepare chlorophyll extract and ana- tively, to prevent cell lysis during filtration, extract whole water
lyze spectrophotometrically. Prepare serial dilutions of the ex- sample.
tract to provide concentrations of approximately 2, 6, 20, and 60 1) Equipment and reagents—Fluorometer equipped with a
␮g chlorophyll a/L. Make fluorometric readings for each solu- high-sensitivity R928 phototube** with output impedance of 36
tion at each sensitivity setting (sliding window orifice): 1⫻, 3⫻, ma/W at 675 nm and a high-sensitivity door. Place neutral
10⫻, and 30⫻. Using the values obtained, derive calibration density filter (40 – 60N) in the rear light path,†† selected to
factors to convert fluorometric readings in each sensitivity level permit reagent blanking on the highest sensitivity scale.
to concentrations of chlorophyll a, as follows: 2) Extraction procedure—Decant 1.5 mL sample into screw-
cap test tube and add 8.5 mL 100% acetone. Mix with vortex
C⬘a mixer and hold in the dark for 6 h at room temperature. Filter
Fs ⫽ R
s through glass fiber filter‡‡ or centrifuge. Measure fluorescence
as described in Section 10200H.3 and estimate concentrations as
where: in ¶ 3c. Because humic substances interfere, if they are present
Fs ⫽ calibration factor for sensitivity setting S, filter a sample portion (see 10200H.1b) and process filtrate with
Rs ⫽ fluorometer reading for sensitivity setting S, and, sample. Subtract filtrate (blank) fluorescence from that of sam-
C⬘a ⫽ concentration of chlorophyll a determined ple.
spectrophotometrically, ␮g/L.

2) Measure sample fluorescence at sensitivity settings that will 4. High-Performance Liquid Chromatographic
provide a midscale reading. (Avoid using the 1⫻ window be- Determination of Algal Chlorophylls and Their Degradation
cause of quenching effects.) Convert fluorescence readings to Products
concentrations of chlorophyll a by multiplying the readings by
the appropriate calibration factor. a. Equipment and reagents: In addition to those listed for
c. Determination of chlorophyll a in the presence of pheophy- pigment extraction, ¶ 1a above:
tin a: This method normally is not applicable to freshwater 1) High-pressure liquid chromatograph capable of a flow rate
samples. See discussion under 10200H and ¶ 2b above. of 2.0 mL/m.
1) Equipment and reagents—In addition to those listed under 2) High-pressure injector valve equipped with a 100-␮L sam-
1a and 2a above, pure chlorophyll a# (or a plankton chlorophyll ple loop.
extract with a spectrophotometric before-and-after acidification 3) Guard column (4.0 ⫻ 0.5 cm, C18 packing material, 3-␮m
ratio of 1.70 containing no chlorophyll b). particle size, or equivalent protection system) for extending life
2) Fluorometric procedure—Calibrate fluorometer as directed of primary column.
in ¶ 3b1). Determine extract fluorescence at each sensitivity 4) Reverse-phase HPLC column.§§
setting before and after acifidication. Calculate calibration fac- 5) Fluorescence detector capable of excitation at 430 ⫾ 30 nm
tors (Fs) and before-and-after acidification fluorescence ratio by and measuring emission at wavelengths greater than 600 nm.
dividing fluorescence reading obtained before acidification by 6) Data recorder device: Strip chart recorder or, preferably, an
the reading obtained after acidification. Avoid readings on the electronic integrator.
1⫻ scale and those outside the range of 20 to 80 fluorometric 7) Syringe, glass, 250-␮L.
units. 8) HPLC eluents: System A (80:15:5; methanol:reagent water:
3) Calculations—Determine the “corrected” chlorophyll a and ion-pairing solution) and System B (80:20; methanol:acetone).
pheophytin a in sample extracts with the following equations:8,24 Use HPLC-grade solvents; measure volumes before mixing.
Filter eluents through a solvent-resistant 0.4-␮m filter before use
r Ve and degas with helium. Prepare the ion-pairing (IP) solution
Chlorophyll a, mg/m3 ⫽ F s (R ⫺ R a)
r⫺1 b Vs from 15 g tetrabutylammonium acetate㛳 㛳 and 77 g ammonium
acetate## made up to 1 L with reagent water.15
r Ve
Pheophytin a, mg/m3 ⫽ F s (rR a ⫺ R b)
r⫺1 Vs
** Hammamatsu Corp., Middlesex, NJ, or equivalent.
where: †† If using Model 10-005, Turner Designs, or equivalent.
Fs ⫽ conversion factor for sensitivity setting S (see ¶ 2b, above), ‡‡ Whatman GF/F or equivalent.
§§ Microsorb C18 column, 10 cm long, 3-␮m particle size, Rainin Co., or equiv-
alent.
㛳 㛳 Fluka Chemical Corp., 980 South Second Street, Ronkonkoma, NY, or equiv-
㛳 Model 10-005, Turner Designs, Sunnyvale, CA or equivalent. alent.
# Purified chlorophyll a, Sigma Chemical Company, St. Louis, MO, or equivalent. ## Sigma Chemical Company, or equivalent.
10-22 BIOLOGICAL EXAMINATION (10000)

9) Calibration standards: Individually dissolve 1 mg each

pure chlorophyll a and b㛳 㛳 in 100 mL 90% acetone. Determine
the exact concentrations spectrophotometrically (⑀664 for chlo-
rophyll a in 90% acetone ⫽ 87.67 L g⫺1 cm⫺1; ⑀647 for chlo-
rophyll b in 90% acetone ⫽ 51.36 L g⫺1 cm⫺1).5 Prepare
pheophytin a ⫹ a⬘ and b ⫹ b⬘ standards from the primary
chlorophyll a and b standards by acidification with hydrochloric
acid; correct respective concentrations for Mg2⫹ loss. Extract
chlorophyll c with 90% acetone from diatoms, purify by thin-
layer chromatography (TLC)25 and calibrate spectrophotometri-
cally (⑀631 for a mixture containing equal amounts of chloro-
phylls c1 and c2 in 90% acetone containing 1% pyridine ⫽ 42.6
L g⫺1 cm⫺1; the absence of this small amount of pyridine is
presumed to cause only small differences in the absorption
properties of chlorophyll c.26 Alternatively, determine the chlo-
rophyll c content of a 90% acetone extract made from diatoms,
spectrophotometrically (chlorophyll c1 ⫹ c2, ␮g/mL ⫽
24.36E630 ⫺ 3.73E664)5 and use as standard. Prepare chlorophyl-
lide a from diatoms,27 purify by TLC25 and calibrate spectro- Figure 10200:11. Reverse-phase HPLC chromatogram for a fivefold
photometrically in 90% acetone (⑀664 for chlorophyllide a ⫽ 128 dilution of EPA sample. Injection volume 100 ␮L; peaks
L g⫺1 cm⫺1).28 Prepare pheophorbide a by acidification of detected by fluorescence spectroscopy (␭ex: 400 – 460 nm;
chlorophyllide a, purify by TLC,25 and calibrate spectrophoto- ␭ex: ⬎600 nm). Peak identities are: 1— chlorophyllide a;
metrically in 90% acetone (⑀665 for pheophorbide a ⫽ 69.8 L g⫺1 2— chlorophyll c; 3—pheophorbide a; 4 — chlorophyll b;
5— chlorophyll a; 6 —pheophytin a; and 7—pheophytin
cm⫺1).28 Standards stored under nitrogen in the dark at ⫺20°C
a⬘. The chlorophyll b degradation products, pheophytin b
are stable for about 1 month. and pheophytin b⬘, were below detection limits. Peak
b. Procedure: identities confirmed by on-line diode array spectroscopy
1) Set up and equilibrate the HPLC system with solvent (350 –550 nm).
System A at a flow rate of 2 mL/min. Adjust fluorometer
sensitivity to provide full-scale reading with the most concen-
trated chlorophyll a standard.
As Fi VE
2) Calibrate HPLC system by preparing working standards Ci ⫽
VI VS
from the primary standards (on day of use). Once retention times
of the standards are determined for a particular system, simplify
standardization by preparing serial dilutions from mixed stan- where:
dards. Prepare separately mixed standards for the chlorophylls Ci ⫽ individual pigment concentration, mg/L,
and chlorophyllide a and for the pheophytins and pheophorbide As ⫽ area of individual pigment peak from sample injection,
a. Mix 1-mL portions of standards with 300 ␮L ion-pairing Fi ⫽ standard response factor (mg pigment/0.1 mL standard
divided by corresponding peak area).
solutions and equilibrate for 5 min before injection (use of
VI ⫽ injection volume (0.1 mL),
ion-pairing agents greatly enhances separation of dephytolated
VE ⫽ extraction volume, mL, and
pigments, chlorophyllide a, chlorophyll c, and pheophorbide a). VS ⫽ sample volume, L.
Prepare blanks by mixing 1 mL 90% acetone with 300 ␮L IP
solution. Rinse syringe twice with 150 ␮L standard and draw 6) This method is designed only for quantification of chloro-
about 250 ␮L standard into syringe for injection. Place syringe phylls and their degradation products. Detect carotenoid pig-
in injector valve, overfilling the 100-␮L sample loop. Construct ments, which also are present in 90% acetone extracts but do not
calibration curves by plotting fluorescence peak areas (or fluoresce, by absorbance spectroscopy (at about 440 nm).21
heights) against standard pigment concentrations. 7) The elution order and approximate retention times for the
3) Prepare samples for injection by mixing a 1-mL portion of major chlorophyll pigments and their degradation products are
the 90% acetone pigment extract with 300 ␮L IP solution. shown in Figure 10200:11. The detection limits (s/n ⫽ 2) vary
4) Use a two-step solvent program to optimize separation of with fluorometer configuration and flow rate; however, they
the chorophylls from their degradation products.15 After injec- range from 10 to 100 pg per injection for most chlorophylls and
tion, change from solvent System A to System B over 5 min and their degradation products.15,21,29 The accuracy of the HPLC
follow with System B for 15 min at a flow rate of 2 mL/min. method depends primarily on purity of pigment standards. Pref-
Re-equilibrate the column with System A for 5 min before the erably measure absorption spectra (350 to 750 nm) of the stan-
next injection for a total analysis time of approximately 25 min. dards and compare with published data. Pigment purity also can
Degas the solvent systems with helium during analysis. Increase be assessed by HPLC analysis, providing there are no co-eluting
lifetime of HPLC column by storing it in 100% methanol be- contaminants with absorption and fluorescence bands overlap-
tween runs. Periodically flush the HPLC system with reagent ping those of the standards. HPLC and spectrophotometrically
water to avoid buildup of ion pairing agents. derived pigment concentrations for available EPA standards
5) Calculate individual pigment concentrations using the fol- agree reasonably well (⫾ 20%) if spectrophotometric results are
lowing formula: corrected for the presence of pheopigments and the HPLC results
PLANKTON (10200)/Chlorophyll 10-23

TABLE 10200:III. EXTINCTION COEFFICIENTS AND CHROMATOGRAPHIC PROPERTIES OF PIGMENTS SEPARATED BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY (CF. FIGURE 10200:12)
Wavelength Retention Absorption Maxima
(solvent) E1cm Ref. Time % c.v. in Eluent*
Pigment Identity nm L g⫺1 cm⫺1 No. min (n ⫽ 3 inj) nm

Chlorophyllide a 664 (90% acetone) 128.0 28 7.8 5.7 nd† nd nd

Chlorophyll c1⫹2 631 (90% acetone) 42.6 26 8.9 0.6 444 576 630
Peridinin 466 (acetone) 134.0 33 10.0 1.2 472
Fucoxanthin 449 (acetone) 160.0 44 11.0 0.9 446 (466)
Neoxanthin 439 (ethanol) 224.3 35 11.5 5.9 416 441 470
Violaxanthin 443 (ethanol) 255.0 35 13.2 2.6 416 440 470
Diadinoxanthin 448 (acetone) 223.0 36 14.6 6.0 422 446 476
Lutein 445 (ethanol) 255.0 35 17.5 0.7 (422) 446 476
Zeaxanthin 450 (ethanol) 254.0 35 18.0 2.2 (428) 454 478
Chlorophyll b 647 (90% acetone) 51.36 5 21.1 1.0 456 596 646
Chlorophyll a 664 (90% acetone) 87.67 5 22.3 0.8 431 618 665
␤,␤-Carotene 453 (90%acetone)‡ 262.0 35 25.4 2.0 427 462 480
*All absorption maxima are from Wright et al.31 except those for chlorophyll c1⫹2 (R.R. Bidigare and M. Latasa, unpublished data).
†Not determined.
‡Because of a potential insolubility problem of ␤,␤-carotene in ethanol, prepare this standard in 90% acetone, not ethanol. It is assumed that the extinction coefficient of
␤,␤-carotene in 90% acetone is the same as that in ethanol.

are expressed as pigment equivalents (e.g., chlorophyll a equiv- 8) HPLC eluents: Eluent A (80:20, v:v; methanol:0.5M am-
alents ⫽ chlorophyllide a ⫹ chlorophyll a ⫹ chlorophyll a⬘, monium acetate, pH 7.2); Eluent B (90:10, v:v; acetonitrile:
provided that the proper molecular weight corrections are ap- water), and Eluent C, ethyl acetate. Use HPLC-grade solvents.
plied).30 Thus, if significant amounts of chlorophyll derivatives Measure volumes before mixing. Filter eluents through a sol-
are present, pigment concentrations determined spectrophoto- vent-resistant 0.4-␮m filter before use and degas with helium.
metrically will be overestimated. The agreement between HPLC 9) Calibration standards: Chlorophylls a and b, and ␤,␤-
and fluorometrically derived results depends on the presence of carotene can be purchased††† as can zeaxanthin and lutein.‡‡‡
accessory chlorophylls b, c, and their derivatives. Triplicate Other pigment standards can be purified from plant extracts by
injections of a fivefold dilution of an EPA sample gave coeffi- thin-layer chromatography (TLC)25 or preparative-scale HPLC.
cients of variation of 7.5% (chlorophyllide a), 9.1% (chlorophyll Determine concentration of all standards using a monochroma-
c), 13.4% (pheophorbide a), 9.6% (chlorophyll b), 0.5% (chlo- tor-based spectrophotometer in the appropriate solvents before
rophyll a), 6.2% (pheophytin a), and 22.9% (pheophytin a⬘), calibration of the HPLC system. The recommended extinction
with an average value of 10% for the seven pigments analyzed. coefficients for most common algal pigments found in freshwa-
ter systems are given in Table 10200:III. Measure absorbance in
5. High-Performance Liquid Chromatographic a 1-cm cuvette at the appropriate wavelength (usually at ␭max)
Determination of Algal Chlorophyll and Carotenoid Pigments and 750 nm (to correct for light scattering). Calculate concen-
trations of standards as follows:
a. Equipment and reagents: In addition to those listed for
pigment extraction, ¶ 1a above: (A ␭max ⫺ A 750nm)
1) High-performance liquid chromatographic pump capable Ci ⫽ ⫻ 1000
E 1cm ⫻ b
of gradient delivery of three different solvents at a flow rate of 1
mL/min. where:
2) High-pressure injector valve equipped with a 200-␮L sam- Ci ⫽ individual pigment concentration, mg/L,
ple loop. A ⫽ absorbance at specific wavelength,
3) Guard column (50 ⫻ 4.6 mm, C18 packing material,*** E1cm ⫽ weight-specific absorption coefficient, L g⫺1 cm⫺1,
5-␮m particle size) for extending life of primary column. b ⫽ pathlength of cuvette, cm, and
4) Reverse-phase HPLC column with endcapping (250 ⫻ 4.6 1000 ⫽ conversion factor, g to mg.
mm, 5-␮m particle size, C18 column***).
5) Variable wavelength or filter absorbance detector with Standards stored under nitrogen in the dark at ⫺20°C are
low-volume flowthrough cell. Detection wavelength is 436 nm. stable for about 1 month.
6) Data recording device: Strip chart recorder or, preferably, b. Procedure:
an electronic integrator or computer equipped with hardware and 1) Set up and equilibrate the HPLC system with Eluent A at a
software for chromatographic data analysis. flow rate of 1 mL/min.
7) Syringe, glass, 500-␮L.
††† Sigma Chemical Co., St. Louis, MO, or equivalent.
‡‡‡ Roth Chemical Co., distributed by Atomergic Chemetals Corp., Farmingdale,
*** Spherisorb ODS-2, Phase Separations Inc., Norwalk, CT, or equivalent. NY, or equivalent.
10-24 BIOLOGICAL EXAMINATION (10000)

Figure 10200:12. Reverse-phase HPLC pigment chromatogram for a mixture of common algal pigments found in freshwater systems. For further data
see Table 10200:III. Sample contained a natural extract with authentic known additions. The small unlabeled peaks are pigment degradation
products.
PLANKTON (10200)/Chlorophyll 10-25

TABLE 10200:IV. HPLC SOLVENT SYSTEM PROGRAM 7) This method is designed for separation of chlorophyll and
carotenoid pigments (Figure 10200:12); however, it also sepa-
Percentage of Eluent
Time Flow Rate rates major chlorophyll breakdown products.
min mL/min A B C Conditions 8) Method precision was assessed by making triplicate injec-
Analysis protocol: tions of a mixture of phytoplankton and plant extracts. Coeffi-
0.0 1.0 100 0 0 Injection cients of variation ranged from 0.6 to 6.0% (Table 10200:III).
2.0 1.0 0 100 0 Linear gradient Using an appropriate internal standard increases precision.
2.6 1.0 0 90 10 Linear gradient Further information on these pigments and on analysis meth-
13.6 1.0 0 65 35 Linear gradient ods is available elsewhere.33–37
18.0 1.0 0 31 69 Linear gradient
23.0 1.0 0 31 69 Hold
25.0 1.0 0 100 0 Linear gradient 6. References
26.0 1.0 100 0 0 Linear gradient
34.0 1.0 100 0 0 Hold
1. ROTT, E. Spectrophotometric and chromatographic chlorophyll anal-
Shutdown protocol:
ysis: comparison of results and discussion of the trichromatic
0.0 1.0 100 0 0 Analysis complete
3.0 1.0 0 100 0 Linear gradient method. Ergebn. Limnol. (Suppl. to Arch. Hydrobiol.) 14:37.
6.0 1.0 0 0 100 Linear gradient 2. MARKER, A.F.H., E.A. NUSCH, H. RAI & B. RIEMANN. 1980. The
16.0 1.0 0 0 100 Washing measurement of photosynthetc pigments in freshwaters and stan-
17.0 0.0 0 0 100 Shutdown dardization of methods: Conclusions and recommendations.
Ergebn. Limnol. (Suppl. to Arch. Hydrobiol.) 14:91.
3. LORENZEN, C.J. 1967. Determination of chlorophyll and pheo-pig-
ments: spectrophotometric equations. Limnol. Oceanogr. 12:343.
2) Calibrate the HPLC using working standards (about 0 to 4. FITZGERALD, G.P. & S.L. FAUST. 1967. A spectrophotometric method
1000 ng/mL) prepared from primary standards on day of use. for the estimation of percentage degradation of chlorophylls to
Mix 1 mL standard with 300 ␮L distilled water, shake, and pheopigments in extracts of algae. Limnol. Oceanogr. 12:335.
equilibrate for 5 min before injection (diluting standards and 5. JEFFREY, S.W. & G.F. HUMPHREY. 1975. New spectrophotometric
sample extracts with water increases affinity of pigments for the equations for determining chlorophylls a, b, and c, in higher plants,
column in the loading step, resulting in an improved separation algae and natural phytoplankton. Biochem. Physiol. Pflanzen 167:
of more polar pigments). Rinse syringe twice with 300 ␮L 191.
standard and draw 500 ␮L standard into syringe for injection. 6. YENTSCH, C.S. & D.W. MENZEL. 1963. A method for the determi-
Place syringe in injector valve, overfilling the 200 ␮L sample nation of phytoplankton chlorophyll and phaeophytin by fluores-
cence. Deep Sea Res. 10:221.
loop 2.5-fold. To check for possible interferences in the extrac-
7. LOFTUS, M.E. & J.H. CARPENTER. 1971. A fluorometric method for
tion solvent and/or filter, prepare a blank by extracting a glass
determining chlorophylls a, b, and c. J. Mar. Res. 29:319.
fiber filter in 90% acetone; mixing 1 mL 90% acetone filter 8. HOLM-HANSEN, O., C.J. LORENZEN, R.W. HOLMES & J.D.H. STRICK-
extract and 300 ␮L distilled water; and injecting into the HPLC LAND. 1965. Fluorometric determination of chlorophyll. J. Cons.
system. Plot absorbance peak areas (or heights) against standard Cons. Perma. Int. Explor. Mer 30:3.
pigment concentrations. Calculate response factors as the slope 9. ABAYCHI, J.K. & J.P. RILEY. 1979. The determination of phytoplank-
of the regression between the weights of the injected standards ton pigments by high-performance liquid chromatography. Anal.
(ng) and the areas of the parent pigment (plus areas of structur- Chim. Acta 107:1.
ally related isomers if present). These isomers contribute to the 10. LORENZEN, C.J. 1966. A method for the continous measurement of in
absorption signal of the standards; disregarding them results in vivo chlorophyll concentration. Deep Sea Res. 13:223.
overestimation of pigments in sample extracts.32 11. JACOBSEN, T.R. 1978. A quantitative method for the separation of
3) Prepare samples for injection by mixing a 1 mL portion of chlorophylls a and b from phytoplankton pigments by high-pressure
liquid chromatography. Mar. Sci. Comm. 4:33.
the 90% acetone pigment extract and 300 ␮L distilled water,
12. BROWN, L.M., B.T. HARGRAVE & M.D. MACKINNON. 1981. Analysis
shake, and equilibrate for 5 min before injection. of chlorophyll a in sediments by high-performance liquid chroma-
4) Following sample injection, use a gradient program to tography. Can. J. Fish. Aquat. Sci. 38:205.
optimize separation of chlorophyll and carotenoid pigments. The 13. GIESKES, W.W. & G.W. KRAAY. 1983. Unknown chlorophyll a
system described in Table 10200:IV has been developed from derivatives in the North Sea and the tropical Atlantic Ocean re-
the original method31 to insure elution of most hydrophobic vealed by HPLC analysis. Limnol. Oceanogr. 28:757.
pigments. Degas solvent system with helium during analysis. 14. GOWEN, R.J., P. TETT & B.J.B. WOOD. 1983. Changes in the major
Periodically flush HPLC system with distilled water to avoid dihydroporphyrin plankton pigments during the spring bloom of phy-
accumulation of ion-pairing reagents. toplankton in two Scoottish sea-lochs. J. Mar. Biol. Assoc. U.K. 63:27.
5) Routinely determine peak identities by comparing retention 15. MANTOURA, R.F.C. & C.A. LLWEWLLYN. 1983. The rapid determi-
times of sample peaks with those of pure standards. Confirm nation of algal chlorophyll and carotenoid pigments and their break-
down products in natural waters by reverse-phase high-performance
peak identities spectrophotometrically by collecting eluting
liquid chromatography. Anal. Chim. Acta 151:297.
peaks from the column outlet (or directly with an on-line diode 16. GIESKES, W.W.C. & G.W. KRAAY. 1984. Phytoplankton, its pig-
array spectrophotometer). Absorption maxima for most common ments, and primary production at a central North Sea station in May,
pigments found in freshwater systems are given in Table 10200: July and September 1981. Neth. J. Sea Res. 18:51.
III. 17. HALLEGRAEFF, G.M. & S.W. JEFFREY. 1985. Description of new
6) Calculate individual pigment concentrations using the for- chlorophyll a alteration products in marine phytoplankton. Deep
mula given in ¶ 4b5) preceding. Sea Res. 32:697.
10-26 BIOLOGICAL EXAMINATION (10000)

18. TREES, C.C., M.C. KENNICUTT II & J.M. BROOKS. 1985. Errors 30. MURRAY, A.P., C.F. GIBBS & A.R. LONGMORE. 1986. Determination
associated with the standard fluorometric determination of chloro- of chlorophyll in marine waters: Intercomparison of a rapid HPLC
phylls and phaeopigments. Mar. Chem. 17:1. method with full HPLC, spectrophotometric and fluorometric meth-
19. ESKINS, K., C.R. SCHOFIELD & H.J. DUTTON. 1977. High-performance ods. Mar. Chem. 19:211.
liquid chromatography of plant pigments. J. Chromatogr. 135:217. 31. WRIGHT, S.W., S.W. JEFFREY, R.F.C. MANTOURA, C.A. LLEWELLYN,
20. WRIGHT, S.W. & J.D. SHEARER. 1984. Rapid extraction and high T. BJORNLAND, D. REPETA & N. WELSCHMEYER. 1991. Improved
performance liquid chromatography of chlorophylls and carotenoids HPLC method for the analysis of chlorophylls and carotenoids from
from marine phytoplankton. J. Chromatogr. 294:281. marine phytoplankton. Mar. Ecol. Prog. Ser. 77:183.
21. BIDIGARE, R.R., M.C. KENNICUTT II & J.M. BROOKS. 1985. Rapid de- 32. BIDIGARE, R.R. 1991. Analysis of algal chlorophylls and carote-
termination of chlorophylls and their degradation products by high-
noids. In D.C. Hurd & D.W. Spencer, eds., Marine Particles: Anal-
performance liquid chromatography. Limnol. Oceanogr. 30:432.
ysis and Characterization. America Geophysical Union, Washing-
22. JEFFREY, S.W. 1974. Profiles of photosynthetic pigments in the
ton, D.C.
ocean using thin-layer chromatography. Mar. Biol. 26:101.
23. PHINNEY, D.A. & C.S. YENTSCH. 1985. A novel phytoplankton chloro- 33. JEFFREY, S.W. & F.T. HAXO. 1968. Photosynthetic pigments of
phyll technique: toward automated analysis. J. Plankton Res. 7:633. symbiotic dinoflagellates (zooxanthallae) from corals and clams.
24. STRICKLAND, J.D.H. & T.R. PARSONS. 1968. A Practical Manual of Biol. Bull. 135:149.
Sea Water Analysis. Fish. Res. Board Can. Bull. No. 167. Queen’s 34. JENSEN, A. 1978. Chlorophylls and carotenoids. In J.A. Helleburst &
Printer, Ottawa, Ont. J.S. Craige, eds., Handbook of Phycological Methods: Physiological
25. JEFFREY, S.W. 1981. An improved thin-layer chromatographic tech- and Biochemical Methods. Cambridge University Press, Cam-
nique for marine phytoplankton pigments. Limnol. Oceanogr. 26:191. bridge, England.
26. JEFFREY, S.W. 1972. Preparation and some properties of crystalline 35. DAVIS, B.H. 1976. Carotenoids. In T.W. Goodwin, ed., Chemistry
chlorophyll c1 and c2 from marine algae. Biochim. Biophys. Acta and Biochemistry of Plant Pigments. Academic Press, New York,
279:15. N.Y.
27. BARRETT, J. & S.W. JEFFREY. 1971. A note on the occurrence of 36. JOHANSEN, J.E., W.A. SVEC & S. LIAAEN-JENSEN. 1974. Carotenoids
chlorophyllase in marine algae. J. Exp. Mar. Biol. Ecol. 7:255. of the Dinophyceae. Phytochemistry 13:2261.
28. LORENZEN, C.J. & J. NEWTON DOWNS. 1986. Specific absorption 37. ARAR, E.J. & G.B. COLLINS. 1992. Method 445.0. In vitro determi-
coefficients of chlorophyllide a and pheophorbide a in 90 percent nation of chlorophyll a and pheophytin a in marine and freshwater
acetone, and comments on the fluorometric determination of chlo- phytoplankton by fluorescence. In Methods for the Determination of
rophyll and pheopigments. Limnol. Oceanogr. 31:449. Chemical Substances in Marine and Estuarine Environmental Sam-
29. SARTORY, D.P. 1985. The determination of algal chlorophyllous ples. U.S. Environmental Protection Agency, Cincinnati, Ohio.
pigments by high performance liquid chromatography and spectro-
photometry. Water Res. 19:605.

10200 I. Determination of Biomass (Standing Crop)

Biomass is a quantitative estimate of the total mass of living 2. Biovolume (Cell Volume)
organisms within a given area or volume. It may include the
mass of a population (species biomass) or of a community Plankton data derived on a volume-per-volume basis often are
(community biomass) but gives no information on community more useful than numbers per milliliter.6 Determine cell volume
structure or function. The most accurate methods for estimation by using the simplest geometric configuration that best fits the
of biomass are dry weight, ash-free dry weight, and volume of shape of the cell being measured (such as sphere, cone, cylin-
living organisms. Indirect methods include estimates of total der).7 Cell sizes of an organism can differ substantially in dif-
carbon, caloric content, nitrogen, lipids, carbohydrates, silica ferent waters and from the same waters at different times during
(diatoms), and chlorophyll (algae). Adenosine triphosphate1 the year; therefore, average measurements from 20 individuals of
(ATP) and deoxyribonucleic acid2,3 (DNA) also have been used each species for each sampling period. Calculate the total bio-
as indirect estimates. All estimates of biomass can be affected by volume of any species by multiplying the average cell volume in
the presence of organic and inorganic detritus; ATP and DNA cubic micrometers by the number per milliliter.
analyses include contributions from the bacterial flora.4 Compute total wet algal volume as:

冘
n
1. Chlorophyll a
Vt ⫽ (Ni ⫻ Vi)
i⫽1
Chlorophyll a is used as an algal biomass indicator.5 Assum-
ing that chlorophyll a constitutes, on the average, 1.5% of the dry
weight of organic matter (ash-free weight) of algae, estimate the where:
algal biomass by multiplying the chlorophyll a content by a Vt ⫽ total plankton cell volume, mm3/L,
Ni ⫽ number of organisms of the ith species/L, and
factor of 67.
Vi ⫽ average volume of cells of ith species, ␮m3.
PLANKTON (10200)/Biomass 10-27

3. Cell Surface Area 4) Freezer (⫺20°C).

5) Boiling water bath.
An estimation of cell surface area is valuable in analyzing 6) Detection instruments designed specifically for measuring
interactions between the cell and surrounding waters. Compute ATP.*
average surface area in square micrometers and multiply by the 7) Microsyringes: 10-, 25-, 50-, 100-, and 250-␮L.
number per milliliter of the species being considered. 8) Reaction cuvettes and vials.
9) Tris buffer (0.02M, pH 7.75): Dissolve 7.5 g tris-hydroxy-
4. Displacement Volume methylaminomethane in 3 L distilled water and adjust to pH 7.75
with 20% HCl. Autoclave 150-mL portions at 115°C for 15 min.
This method8 measures an equivalent volume of liquid that is 10) Luciferin-luciferase enzyme preparation:† Rehydrate fro-
displaced by the sample. Displacement volume may be deter- zen (⫺20°C) lyophilized extracts of firefly lanterns with Tris
mined by several methods; for simple, direct measurement pro- buffer as directed by the supplier; let stand at room temperature
ceed as follows: Place sample in sieve of mesh size equal to or 2 to 3 h, then centrifuge at 300 ⫻ g for 1 min and decant the
smaller than net used in capture; let sample drain and transfer to supernatant into a clean, dry test tube; let stand at room temper-
a measured volume of water in a graduated cylinder; measure the ature for 1 h.
new volume containing sample plus known volume. The dis- 11) Purified ATP standard: Dissolve 12.3 mg disodium ATP
placement volume equals the new volume minus original mea- in 1 L distilled water and dilute 1.0 mL to 100 mL with Tris
sured volume of water. buffer; 0.2 mL ⫽ 20 ng ATP.
b. Procedure:
5. Gravimetric Methods 1) Calibration—To determine the calibration factor (F), pre-
pare a series of dilutions of purified ATP standard and record the
The biomass of the plankton community can be estimated light emission from several portions of each concentration of
from gravimetric determinations, although silt and organic de- standard. Correct mean area of standards by subtracting peak
tritus interfere. Determine dry weight by placing 100 mg wet reading or mean area of several blanks using 0.2 mL Tris buffer.
concentrated sample in a clean, ignited, and tared porcelain Calculate calibration factor FS as:
crucible and dry at 105°C for 24 h. Alternatively, filter a known
volume of sample through 0.45-␮m-pore-diam membrane or a C
prerinsed, dried, and preweighed glass-fiber filter. (Note that the FS ⫽
AS
small sample used in direct filtration may lead to error if not
handled properly.) Cool sample in a desiccator and weigh. Ob- where:
tain ash weight by igniting the dried sample at 500°C for 1 h. FS ⫽ calibration factor at sensitivity S,
Cool, rewet ash with distilled water, and bring to constant weight AS ⫽ peak reading or mean area under standard ATP curve
at 105°C. The ash is rewetted to restore water of hydration of corrected for blank, and
clays and other minerals; this may amount to as much as 10% of C ⫽ concentration of ATP in standard solution, ng/mL.
weight lost during incineration.9 The ash-free dry weight is the
difference between the dry weight and the weight of the ash 2) Sample analysis—Collect a 1- to 2-L sample in a clean,
residue after ashing. The ash-free dry weight is preferred to dry sterile sampler. Pass through a 250-␮m net to remove large
weight to compare mixed assemblages. The ash content may zooplankton10 and filter through a 47-mm 0.45-␮m-porosity
constitute 50% or more of the dry weight in phytoplankton filter by applying a vacuum of about 30 kPa. (IMPORTANT: Break
having inorganic structures, such as the diatoms. In other forms the suction before the last film of water is pulled through the
the ash content is only about 5% of dry weight. filter.) Quickly place filter in a small beaker. Immediately cover
filter with 3 mL boiling Tris buffer, using an automatic pipet.
6. Adenosine Triphosphate (ATP) Place beaker in boiling water bath for 5 min and, with a Pasteur
pipet, transfer extract to a clean, dry, calibrated test tube. Rinse
Methods of measuring adenosine triphosphate (ATP) in plank- filter and beaker with 2 mL boiling Tris buffer; combine extracts,
ton provide the only means of determining the total viable record volume, bring volume up to 5 mL with Tris buffer, cover
plankton biomass. ATP occurs in all plants and animals, but only tubes with parafilm and, if samples cannot be analyzed imme-
in living cells; it is not associated with nonliving particulate diately, freeze at ⫺25°C. Extracts may be stored for many
material. The ratio of ATP to biomass varies from species to months in a freezer. Prepare at least triplicate extracts of each
species, but appears to be constant enough to permit reliable sample.
estimates of biomass from ATP measurements.10 The method is The analytical procedure depends on detection equipment
simple and relatively inexpensive and the instrumentation is used. If a scintillation counter is used, pipet 0.2 mL enzyme
stable and reliable. The method also has many potential appli- preparation into a glass vial. Measure the light emission of the
cations in entrainment and bioassay work, especially plankton enzyme preparation (blank) for 2 to 3 min at sensitivity settings
mortality studies. near that anticipated for the sample. Add 0.2 mL sample extract
a. Equipment and reagents: to the vial, record the time, and swirl. Start recording light output
1) Glassware: clean, sterile, dry borosilicate glass flasks, 10 s after combining ATP extract and enzyme preparation;
beakers, and pipets.
2) Filters: 47-mm-diam, 0.45-␮m-porosity membrane filters. * Beckman, JRB, Turner Designs, or equivalent.
3) Filtration equipment. † Dupont, Sigma Chemical, or equivalent.
10-28 BIOLOGICAL EXAMINATION (10000)

record output for 2 to 3 min, using the same time period for all 2. HOLM-HANSEN, O., W.H. SUTCLIFFE, JR. & J. SHARP. 1968. Measure-
samples. Determine the mean of areas under the curves obtained ment of deoxyribonucleic acid in the ocean and its ecological
and correct by subtracting mean of areas under the curves significance. Limnol. Oceanogr. 13:507.
obtained from blanks prepared as directed in Strickland and 3. HOLM-HANSEN, O. 1969. Determination of microbial biomass in
ocean profiles. Limnol. Oceanogr. 14:740.
Parsons.11
4. PAERL, H.W., M.M. TILZER & C.R. GOLDMAN. 1976. Chlorophyll a
c. Calculations: Calculate concentration of ATP as: vs. ATP as algal biomass indicators in lakes. J. Phycol. 12:242.
5. CREITZ, G.I. & F.A. RICHARDS. 1955. The estimation and character-
Ac ⫻ Ve ⫻ Fs ization of plankton populations by pigment analysis. J. Mar. Res. 14:211.
ATP, ng/L ⫽
Vs 6. KUTKUHN, J.H. 1958. Notes on the precision of numerical and
volumetric plankton estimates from small sample concentrations.
where: Limnol. Oceanogr. 3:69.
Ac ⫽ mean corrected area under extract curves, 7. VOLLENWEIDER, R.A. 1969. A Manual on Methods for Measuring
Primary Production in Aquatic Environments. IBP Handbook No.
Ve ⫽ extract volume, mL,
12. Blackwell Scientific Publ., Oxford, England.
Vs ⫽ volume of sample, L, and
8. JACOBS, F. & G.C. GRANT. 1978. Guidelines for zooplankton sam-
Fs ⫽ calibration factor.
pling in quantitative baseline and monitoring programs. EPA-600/
3-78-026, U.S. Environmental Protection Agency.
If an ATP content of 2.4 ␮g ATP/mg dry weight organic 9. NELSON, D.J. & D.C. SCOTT. 1962. Role of detritus in the produc-
matter is assumed,12 total living plankton biomass (B), as dry tivity of a rock-outcrop community in a Piedmont stream. Limnol.
weight organic matter, is given as: Oceanogr. 7:396.
10. RUDD, J.W.M. & R.D. HAMILTON. 1973. Measurement of adenosine
triphosphate (ATP) in two precambrian shield lakes of northwestern
ATP
B, mg/L ⫽ Ontario. J. Fish. Res. Board Can. 30:1537.
(2.4)(1000) 11. STRICKLAND, J.D.H. & T.R. PARSONS. 1968. A Practical Manual of
Sea Water Analysts. Fish. Res. Board Can. Bull. No. 167. Queen’s
7. References Printer, Ottawa, Ont.
12. WEBER, C.I. 1973. Recent developments in the measurement of the
1. HOLM-HANSEN, O. & C.R. BOOTH. 1966. The measurement of aden- response of plankton and periphyton to changes in their environ-
osine triphosphate in the ocean and its ecological significance. ment. In G. Glass, ed. Bioassay Techniques and Environmental
Limnol. Oceanogr. 11:510. Chemistry. Ann Arbor Science Publ., Ann Arbor, Mich.

10200 J. Metabolic Rate Measurements

The physiological condition and the spectrum of biological 2. Productivity, Oxygen Method
interactions of the aquatic community must be considered for
evaluation of the state of natural waters. Earlier, numbers, spe- Productivity is defined as the rate at which inorganic carbon
cies composition, and biomass were the prime considerations. is converted to an organic form. Chlorophyll-bearing organ-
Recognition of the limitations of this approach led to the mea- isms (phytoplankton, periphyton, macrophytes) serve as pri-
surement of rates of metabolic processes such as photosynthesis mary producers in the aquatic food chain. Photosynthesis
(productivity), nitrogen fixation, respiration, and electron trans- ultimately results in the formation of a wide range of organic
port. These provide a better understanding of the complex nature compounds, release of oxygen, and reduction of carbon diox-
of the aquatic ecosystem. An indication of photosynthetic effi- ide (CO2) in the surrounding waters. Primary productivity6
ciency can be determined by the productivity index (mg C can be determined by measuring the changes in oxygen and
fixed/unit chlorophyll a).1 CO2 concentrations.7 In poorly buffered waters, pH can be a
sensitive property for detecting variations in the system. As
CO2 is removed during photosynthesis, the pH rises. This
1. Nitrogen Fixation
shift can be used to estimate both photosynthesis and respi-
ration.8 The sea and many fresh waters are too highly buffered
The ability of an organism to fix nitrogen is a great compet-
to make this useful, but it has been applied successfully to
itive advantage and plays a major role in population dynamics.
productivity studies in some lake waters.
Two reliable methods for estimating nitrogen fixation rates in the
Two methods of measuring the rate of carbon uptake and net
laboratory are the 15N isotope tracer method2,3 and the acetylene
reduction method.4 Because the rate of nitrogen fixation varies photosynthesis in situ are the oxygen method9 and the carbon 14
greatly with different organisms and with the concentration of method.10 In both methods, clear (light) and darkened (dark)
combined nitrogen, nitrogen fixation rates cannot be used to bottles are filled with water samples and suspended at regular
estimate biomass of nitrogen-fixing organisms. However, the depth intervals for an incubation period of several hours or
acetylene reduction method is useful in measuring nitrogen samples are incubated under controlled conditions in environ-
budgets and in algal assay work.5 mental growth chambers in the laboratory.
PLANKTON (10200)/Metabolic Rate Measurements 10-29

The basic reactions in algal photosynthesis involve uptake of acid (H2SO4) or check with an oxygen probe. Analyses may be
inorganic carbon and release of oxygen, summarized by the delayed several hours if necessary, if samples are fixed or iced
relationship: and stored in the dark.
6) Suspend duplicate paired clear and darkened bottles at the
depth from which the samples were taken and incubate for at
CO2 ⫹ H2O 3 (CH2O)x ⫹ O2
least 2 h, but never longer than it takes for oxygen-gas bubbles
to form in the clear bottles or DO to be depleted in the dark
The chief advantages of the oxygen method are that it pro- bottles.
vides estimates of gross and net productivity and respiration 7) At the end of the exposure period, immediately determine
and that analyses can be performed with inexpensive labora- DO as described above.
tory equipment and common reagents. The dissolved oxygen c. Calculations: The increase in oxygen concentration in the
(DO) concentration is determined at the beginning and end of light bottle during incubation is a measure of net production
the incubation period. Productivity is calculated on the as- which, because of the concurrent use of oxygen in respiration, is
sumption that one atom of carbon is assimilated for each somewhat less than the total (or gross) production. The loss of
molecule of oxygen released. oxygen in the dark bottle is used as an estimate of total plankton
a. Equipment: respiration. Thus:
1) BOD bottles, numbered, 300-mL, clear and opaque boro-
silicate glass, with ground glass stopper and flared mouth, for Net photosynthesis ⫽ light bottle DO ⫺ initial DO
sample incubation. Acid-clean the bottles, rinse thoroughly with
distilled water, and just before use, rinse with water being tested. Respiration ⫽ initial DO ⫺ dark bottle DO
Do not use phosphorus-containing detergents.
Gross photosynthesis ⫽ light bottle DO ⫺ dark bottle DO
If suitable opaque bottles are not available, make clear BOD
bottles opaque by painting them black and wrapping with black
Average results from duplicates.
waterproof tape. As a further precaution, wrap entire bottle in
1) Calculate the gross or net production for each incubation
aluminum foil or place in light-excluding container during incu-
depth and plot:
bation.
2) Supporting line or rack that does not shade suspended
bottles. mg carbon fixed/m3 ⫽ mg oxygen released/L ⫻ 12/32 ⫻ 1000 L/m3 ⫼ K
3) Nonmetallic opaque acrylic Van Dorn sampler or equiva-
lent, of 3- to 5-L capacity. where K is the photosynthetic quotient (PQ), ranging from 1 to 2,
4) Equipment and reagents for dissolved oxygen determina- depending on the nitrogen supply.11,12
tions: See Section 4500-O. Use the factor 12/32 to convert oxygen to carbon; under ideal
5) Pyrheliometer. conditions 1 mole of O2 (32 g) is released for each mole of
6) Submarine photometer. carbon (12 g) fixed.
7) Thermometer. 2) Productivity is defined as the rate of production and gen-
b. Procedure: erally is reported in grams carbon fixed per square meter per day.
1) Obtain a profile of the input of solar radiation for the Determine the productivity of a vertical column of water 1 m
photoperiod with a pyrheliometer. square by plotting productivity for each exposure depth and
2) Determine depth of euphotic zone (the region that receives graphically integrating the area under the curve.
1% or more of surface illumination) with a submarine photom- 3) Using the solar radiation profile and photosynthesis rate
eter. Select depth intervals for bottle placement. The photosyn- during incubation adjust the data to represent phytoplankton
thesis-depth curve will be approximated closely by placing sam- productivity for the entire photoperiod. Because photosynthetic
ples at intervals equal to one-tenth the depth of the euphotic rates vary widely during the daily cycle,13,14 do not attempt to
zone. Estimate productivity in relatively shallow water with convert data to other test circumstances.
fewer depth intervals.
3) Measure oxygen concentration with probe or by titration 3. Productivity, Carbon 14 Method
and temperature and salinity to determine whether water is
supersaturated with respect to oxygen (see Table 4500-O:I). If A solution of radioactive carbonate (14CO32–) is added to light
water is supersaturated, bubble nitrogen gas through sample to and dark bottles that have been filled with sample as described
lower initial oxygen concentration to less than 80% saturation. for the oxygen method. After incubation in situ, collect the
4) Keep samples out of direct sunlight during handling. Intro- plankton on a membrane filter, treat with hydrochloric acid
duce samples taken from each preselected depth into duplicate (HCl) fumes to remove inorganic carbon 14, and assay for
clear, darkened, and initial-analysis bottles. Insert delivery tube radioactivity. The quantity of carbon fixed is proportional to the
of sampler to bottom of sample bottle and fill so that three fraction of radioactive carbon assimilated.
volumes of water are allowed to overflow. Remove tube slowly This procedure differs from the oxygen method in that it
and close bottle. Use water from the same grab sample to fill a affords a direct measurement of carbon uptake and measures
“set” (one light, one dark, and one initial bottle). only net photosynthesis.15 It is basically more sensitive than the
5) Immediately treat (fix) samples taken for the chemical oxygen method, but fails to account for organic materials that
determination of initial dissolved oxygen (see Section 4500-O) leach from cells16,17 during incubation.
with manganous sulfate (MnSO4), alkaline iodide, and sulfuric a. Equipment and reagents:
10-30 BIOLOGICAL EXAMINATION (10000)

1) Pyrheliometer. bation period to entire photoperiod. For incubation procedure,

2) Submarine photometer. see ¶ 2b6) above.
3) BOD bottles and supporting apparatus: See ¶s 2a1) and 2), 5) After incubating remove sample bottles and immediately
above. place in the dark. Filter unpreserved samples without delay.
4) Membrane-filtering device and 25-mm filters with pore Avoid sample preservation to avoid lysing cells or determine
diameters of 0.22, 0.30, 0.45, 0.80, and 1.2 ␮m. extracellular products.
5) Counting equipment for measuring radioactivity: Scaler 6) Filter two portions of each sample through a membrane
with end-window tube, gas flow meter, or liquid scintillation filter, taking care that the largest pore size is consistent with
counter (see Section 7030B). The thin-window tube is the least quantitative retention of plankton. Although the 0.45-␮m pore
expensive detector and, when used with a small scaler, provides filter usually is adequate, determine the efficiency of sample
acceptable data at modest cost. retention immediately before analysis, with a wide range of pore
6) Fuming chamber: Use a glass desiccator with a depth of sizes.23,24 Apply approximately 30 kPa of vacuum during filtra-
about 1.4 cm conc HCl in desiccant chamber. The fuming tion. Excess vacuum may cause extensive cell rupture and loss of
chamber is recommended for filter decontamination.18,19 radioactivity through the membrane.25 Use maximum sample
7) Syringe or pipet, nonmetallic. volume consistent with rapid filtration (1 to 2 min), but do not
8) Chemical reagents: See Sections 4500-CO2 (Carbon Diox-
clog filter.
ide) and 2320 (Alkalinity).
7) Place membranes in HCl fumes for 20 min. Count filters as
9) Radioactive carbonate solutions:
soon as possible, although extended storage in a desiccator is
a) Sodium chloride dilution solution, 5% NaCl (w/v): Add
acceptable.
0.3 g sodium carbonate (Na2CO3) and one pellet sodium hydrox-
8) Determine radioactivity by counting with an end-window
ide (NaOH) per liter. Use for marine studies only.
b) Carrier-free radioactive carbonate solution, commercially tube, windowless gas flow detector, or liquid scintillation
available in sealed vials having approximately 5 ␮Ci 14C/mL. counter.
Confirm absence of suspended and dissolved toxic metals20 or 9) Determine counting geometry of thin-window and window-
filter and pass through an ion-exchange column.* less gas flow detectors.26 Using three ampules of carbon 14,
c) Working solutions with activities of 1, 5, and 25 ␮Ci 14C/2 prepare a series of barium carbonate (BaCO3) precipitates on
mL. For studies of fresh water use carrier-free radioactive car- tared 0.45-␮m membrane filters as directed below. The precip-
bonate and for studies of marine water prepare by diluting itates will contain the same amount of carbon 14 activity but will
carrier-free radioactive carbonate solution with NaCl dilution have different thicknesses ranging from 0.5 to 6.0 mg/cm2.
solution. Dilute each ampule to 500 mL with a solution of 1.36 g
d) Stock ampules: Prepare ampules containing 2 mL of re- Na2CO3/L CO2-free distilled water. Pipet 0.5-mL portions into
quired working solution. Fill ampules and autoclave sealed am- each of seven conical flasks containing 0, 0.5, 1.5, 2.5, 3.5, 4.5,
pules at 121°C for 20 min.21 and 5.5 mL, respectively, of a solution of 1.36 g Na2CO3 /L
b. Procedure: CO2-free distilled water. Add, respectively, 0.3, 0.6, 1.2, 1.8, 2.4,
1) Obtain a record of incident solar radiation for the photope- 3.0, and 3.6 mL 1.04% barium chloride (BaCl2) solution. Let
riod with a pyrheliometer. BaCO3 precipitate stand 2 h with gentle swirling every half hour.
2) Determine depth intervals for sampling and incubation as Collect each precipitate on a filter (using an apparatus with a
described above. filtration area comparable to that of the samples). With suction,
3) Use duplicate light and dark bottles at each depth. Also use dry filters without washing; place in a desiccator for 24 h, weigh,
dark bottles or bottles harvested at time zero. Fill bottles with and count. The counting rate increases exponentially with de-
sample, add 2 mL radioactive carbonate solution (using a non- creasing precipitate thickness. Extrapolate graphically (or math-
metallic pipet) to the bottom of each bottle, and mix thoroughly ematically) to zero precipitate thickness and multiply the zero-
by repeated inversion. The concentration of carbon 14 should be thickness counting rate by 1000 to correct for ampule dilution.
approximately 10 ␮Ci/L in relatively productive waters, to 100 This represents the amount of activity added to each sample
␮Ci/L, or higher, in oliogotrophic (open ocean) waters. To bottle used to determine fraction of carbon 14 taken up in light
obtain statistical significance, have at least 1000 cpm in the and dark bottles.
filtered sample. Take duplicate samples at each depth to deter- c. Calculations:
mine initial concentration of inorganic carbon (CO2, HCO3– and 1) Subtract the mean dark-bottle or time-zero sample count
CO32–) available for photosynthesis (see Section 4500-CO2). For from the mean light-bottle counts for each replicate pair.
estuarine and marine samples, estimate total inorganic carbon 2) Determine the total dissolved inorganic carbon available for
concentrations with a simple titration procedure22 and make
photosynthesis (carbonate, bicarbonate, and free CO2) from pH
initial temperature, salinity, and pH measurements.
and alkalinity measurements; make direct measurement of total
4) Incubate samples for up to 4 h. If measurements are
CO2 according to Section 4500-CO2 or the methods described in
required for the entire photoperiod, overlap 4-h periods from
the literature.27–30
dawn until dusk. A 4-h incubation period may be sufficient
3) Determine quantity of carbon fixed by using the following
provided energy input is used as the basis for integrating incu-
relationship:

* Chelex 100 or equivalent.

PERIPHYTON (10300)/Metabolic Rate Measurements 10-31

counting rate of filtered sample 12. DAVIES, J.M. & P.J. LEB. WILLIAMS. 1984. Verification of 14C and O2
mg carbon fixed/L ⫽ derived primary organic production using an enclosed system. J.
total activity added to sample
Plankton Res. 6:457.
300 13. RYTHER, J.H. 1956. Photosynthesis in the ocean as a function of light
⫻ ⫻ mg/L initial inorganic carbon ⫻ 1.064† intensity. Limnol. Oceanogr. 1:61.
volume filtered
14. FEE, E.J. 1969. A numerical model for the estimation of photosyn-
thetic production, integrated over time and depth, in natural waters.
4) Integrate productivity for the entire depth of euphotic zone Limnol. Oceanogr. 14:906.
and express as grams carbon fixed per square meter per day [see 15. STEEMAN-NEILSEN, E. 1964. Recent advances in measuring and un-
¶ 2c2) above]. derstanding marine primary production. J. Ecol. 52(Suppl.):119.
5) Using the solar radiation records and photosynthesis rates 16. ALLEN, M.B. 1956. Excretion of organic compounds by Chlamydo-
during incubation, adjust data to represent phytoplankton pro- monas. Arch. Mikrobiol. 24:163.
ductivity for the entire photoperiod. If samples were incubated 17. FOGG, G.E. & W.D. WATT. 1965. The kinetics of release of extra-
for less than the full photoperiod, apply a correction factor. cellular products of photosynthesis by phytoplankton. In C.R. Gold-
man, ed. Primary Productivity in Aquatic Environments. Suppl. 18,
4. References Univ. California Press, Berkeley.
18. WETZEL, R.G. 1965. Necessity for decontamination of filters in C14
measured rates of photosynthesis in fresh waters. Ecology 46:540.
1. GUNDERSEN, K. 1973. In-situ determination of primary production
19. MCALLISTER, C.D. 1961. Decontamination of filters in the C14
by means of the new incubator, ISIS. Helgolander wiss. Meere-
method of measuring marine photosynthesis. Limnol. Oceanogr.
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† Correction for isotope effect.