WATER POLLUTION SURVEILLANCE SYSTEM
APPLICATIONS AND DEVELOPMENT REPODT
No. 19
METHODS OF COLLECTION AND ANALYSIS OF
PLANKTON AND FERIPHYTON SAMPLES IN
THE WATER POLLUTION SURVEILLANCE S2STEM
Cornelius 1. Weber, Ph.D.
JUly 1966
DIVISION OF POLLUTION SURVEILLANCE
FEDERAL WATER POLLUTION CONTROL ADMINISTRATION
DEPARTMENT OF THE INTERIOR
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600R66115
PREFACE
Dr. Weber is in charge of plankton and periphyton studies and
serves as Assistant Chief of Aquatic Biology in the Water Pollution
Surveillance System Laboratories at Cincinnati, Ohio. Since his
association with this activity in September 1963, he has conducted
a number of evaluations of methods and techniques. In addition, he
has developed sampling and analysis procedures for periphyton.
Studies of the periphyton can be especially useful in detecting
influences of specific pollution sources when samplers are appro-
priately located. The Aquatic Biology program is directed by
Mr. Joseph B. Anderson.
The methods for analysis of plankton samples described in this
report are essentially those developed by Dr. Louis G. Williams
while he was in charge of the plankton program during the period
October 1958 - December 1962. A significant change subsequently
introduced in plankton analysis, however, was the use of centrifu-
gation rather than settling for the concentration of diatoms.
A. W. Breidenbach, Ph.D.
Assistant Chief for Laboratories
Division of Pollution Surveillance
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Table of Contents
Page
I. Plankton
A. Collection 1
B. Preservation 1
C. Sedgwick-Rafter Pnytoplankton Analysis 3
D. Diatom Species Proportional Analysis 6
E. Zooplankton Analysis 13
II. Periphyton
A. Collection 16
B. Preservation 16
C. Sample Preparation 16
D. SedgvrLck-Rafter Cell Analysis 18
E. Diatom Species Proportional Analysis 19
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Figures
Page
1. Plankton Sample Bottles and Shipping Containers. 2
2. Filling the Sedgwick-Rafter Cell. k
3. Sedgwlck-Rafter Strip Count. 5
k. Diatom Slide. 9
5. Settling Tube. 11
6. Lower Portion of a Settling Tube. 12
7. Zooplankton Counting Chamber. 1^
8. Periphyton Sampler. 17
9. Periphyton Sample Bottle. 18
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METHODS OF COLLECTION AMD ANALYSIS OF
PLANKTON AND PERIPHYTON SAMPLES IN
THE WATER POLLUTION SURVEILLANCE SYSTEM
I. Plankton
A. Collection
Plankton samples are obtained from water plant intakes
or directly from lakes or rivers at a depth of 2 to 15 feet.
The sample volume varies from 1 to 3 liters, depending on the
types of analyses to be performed. One liter is sufficient for
a phytoplankton Sedgwick-Rafter count and diatom species anal-
ysis; a 3-liter sample is collected if a zooplankton count is
also to be made. The narrow-mouth polyethylene sample bottles
are shipped in individual, cushioned, fiberboard cartons
(Figure l), and contain MERTKEOLATE preservative when mailed
to the station. The bottles are accompanied by a sampling
date reminder, and a tag (Appendix) for the sampling data.
B. Preservation
The MERTHIOLATE preservative stock solution is prepared by
dissolving the following in 1 liter of distilled water:
1.0 gram of MERTHIOLATE (sodium ethyl-mercury thiosalicylate)
1,0 ml of aqueous saturated Iodine-KI solution prepared by
dissolving 60 grams of KI and ko grams of l£ in 1 liter
of distilled water
1.5 grams of Borax (sodium borate)
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-2-
Figure 1. Plankton Sample Bottles and Shipping Containers,
To each plankton sample bottle shipped from our laboratory
sufficient volume of stock solution is added to provide 36 mg
of MERTHIOLATE, 5^ mg of Borax, and 1.3 mg of Iodine per liter
of water when the bottle is filled with sample. This preser-
vative effects excellent color retention and causes no morpho-
logical distortion. Although sterility is not achieved at this
concentration of MERTMOLATE, samples may be stored on the shelf
at least 1 year without deterioration. Phytoplankton growth is
arrested at MEIRTHIOLATE concentrations as low as 2 mg per liter,
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-3-
but gradual bacterial deterioration of the plankton occurs
at less than 10 ppm. The cost of preserving a 3-liter sample
is approximately $0.02.
C. Sedgwick-Rafter Phytoplankton Analysis
The plankton sample is mixed by inverting the sample
bottle no fewer than seven times, and a 50- to 100-ml volume
is poured immediately into a small beaker. The contents of
the beaker are well mixed by repeatedly filling and discharging
a 1-ml pipette. Then, without delay, the pipette is filled
with sample, and the liquid is directed diagonally across the
bottom of a Sedgwick-Rafter cell. (One-half of the chamber is
filled from each of the opposite corners - see Figure 2.) As
the chamber fills, the cover glass rotates on the water film
and becomes aligned with the chamber. Excess water in and
around the chamber is removed with a blotter. After it is
filled, the counting chamber is placed on the microscope stage
and allowed to stand 15 minutes to permit the algae to settle
to the bottom.
If the phytoplankton are obscured by silt, a 1-ml aliquot
of sample is diluted 5 to 10 times with tap water and the cell
is refilled.
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-4-
Figure 2. Fining the Sedgwick-Rafter Cell.
The count is made by scanning two strips across the cell
(Figure 3) at 200X, each strip being the width of a Whipple
grid (approximately 0.45 mm). Two longitudinal strips include
o
an area approximately twice 0.45 X 50 mm, or 45 mm . Since
the chamber is 1 mm deep, the total volume examined would be
0.045 ml. The bottom of the cell is divided into five sections
by transverse lines used as reference marks when scanning.
As the non-diatoms are counted, they are identified to
species, if possible, and tallied on a bench sheet (Appendix)
in one of the following categories: coccoid blue-green,
filamentous blue-green, coccoid green, filamentous green,
green flagellate, or other flagellated algae. Each solitary
cell, or natural group (colony) of cells, is tallied as one
unit. If, during a count, 100 or more of a given alga are
tallied in the first section of t he Sedgwick-Rafter cell
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-5-
(a tenth of the total scanned area), the tally for this
organism is immediately converted to units per ml and the
alga is disregarded for the rest of the count. This pro-
cedure is followed whenever 100 or more of any organism are
tallied before the count is nine-tenths complete.
A
!i
i
!
i
•—
*^*
—— .
-. —
strips
scanned
Figure 3. Sedgwick-Rafter Strip Count.
A cell count (not a unit count) is made of the diatoms,
which are tallied as live Gentries, centric shells (empty
frustules), live pennates, or pennate shells (empty frustules).
In practice, frustules containing any part of a protoplast are
tallied as live.
If a sample contains organisms so small they are difficult
to identify at 200X, a 10-ml aliquot is centrifuged and a wet
mount is examined at 970X. Those forms that cannot be identi-
fied with certainty are arbitrarily assigned to the category
considered most appropriate by the examining biologist.
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-6-
D. Diatom Species Proportional Analysis
Diatom species proportional counts are made from perma-
nent slides prepared from plankton concentrates obtained by
centrifuging aliquots of the samples. Routinely, a 100-ml
aliquot of a thoroughly mixed sample is centrifuged 20 minutes
at 1000 G, and the supernatant water is decanted with a suction
tube. Tests have shown that the diatoms are quantitatively
removed from the aliquot by centrifugation. The plankton
concentrate is poured into a disposable 3-dram vial, and the
station number, name, and date are written on the side of the
vial with a black, felt, marking pen. The vial is then
allowed to stand at least 2k hours before further processing.
All but a few mllliliters of water are then withdrawn
from the vial with a suction tube. If the water contains more
than 1 gm of dissolved solids per liter, as in the case of
brackish water or marine samples, the salt crystals will obscure
the diatom frustules on the finished slides. In this case, the
concentration of salts is reduced by refilling the vial with
distilled water, resuspending the plankton, and allowing the
vial to stand 2k hours before removing the supernatant liquid.
The dilution is repeated several times if necessary.
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-7-
The diatom slides are prepared as follows:
1. The plankton concentrate In a vial is thoroughly mixed
with a disposable pipette, and several drops are delivered to a
No. 1 circular, 18-mm coverglass. Twenty to 30 samples are
usually processed at one time by placing the coverglasses on a
piece of sheet metal, 5 X 10 X 1/8 inches.
2c The samples are dried on a hotplate at 95°C. (Caution:
overheating may cause splattering and cross-contamination of the
samples.)
3. When the material has dried, the coverglasses are
examined to determine if there is sufficient material for a
diatom count.
k-. Steps No. 1 and 2 are repeated one or more times,
depending on the density of plankton and sediment in the vial.
5« The metal plate bearing the coverglass is then heated
at approximately 1000°F for 30 minutes, (it is best to have
two hotplates; a low-temperature plate for drying, and a high-
temperature plate for incinerating.)
6. Using a No. 3 pencil, the frosted end of a 25- X 75-mm
microscope slide is labeled with the name of the river or lake,
the station name and number, and the sampling date (Figure 4).
7» The labeled slide is then placed on a moderately warm
hotplate (250°F), a drop of Hyrax mounting medium (R. I. 1.65)
is placed in the center, and the slide is heated until the hyrax
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-8-
solvent (xylene) is driven off. When the solvent has
evaporated, the slide is ready to receive the coverglass.
One can determine when the solvent is gone by periodically
touching a dissection needle to the Hyrax on the slide and
allowing the needle to cool. The Hyrax will become hard
and brittle upon cooling. (The same hotplate used to dry
the plankton concentrate on the coverglass is used to
prepare the Hyrax on the slide.)
8. Grains of sand or other large objects on the cover-
glass should be removed with a dissection needle. The oil
Immersion objective has a very small working distance, and
the slide may be unusable if this material is not removed.
9. While the coverglass and slide are still hot, the
coverglass is grasped with a tweezer, inverted, and placed
on the drop of melted Hyrax on the slide. Slight pressure
is applied to the coverglass with a cylindrical object
(e.g. pencil eraser), and the coverglass is centered on the
slide. It may be necessary to add Hyrax at the margin of the
coverglass.
10. Some additional bubbles of solvent vapor may appear
under the coverglass when it is placed on the slide. When
the bubbling ceases, the slide is removed from the hotplate
and placed on a firm, flat surface. Pressure is immediately
applied to the coverglass as described in step No. 9 and
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-9-
malntained until the Hyrax cools and hardens (about 5 seconds).
Bubbles in the Hyrax are pressed out by moving the pencil
eraser around the edge of the coverglass.
11. A protective coating of clear lacquer is sprayed on
the frosted end of the slide.
12. The excess Hyrax is scraped from around the cover-
glass.
To begin the diatom count, the slide is scanned to locate
an area that is relatively free of silt and contains a moderate
density of diatoms. Lateral strips the vidth of the Whipple
grid are then examined (Figure k), and a.n diatoms within the
borders of the grid are counted and identified to species
(see bench sheet in Appendix).
-
Figure 4. Diatom Slide.
If, before the count is completed, the lateral movement of
the slide brings the grid image to the edge of the coverglass
or to an area of dense sediment, the slide is shifted up or
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-10-
down and the count is continued in another strip. Small
cell fragments are ignored.
In a typical diatom analysis, 200 to 300 diatom cells
are identified and tallied on the bench sheet. However, if
the slide has a scarcity of diatoms, dictated by the lack
of material in the sample, the analysis is limited to the
number of cells encountered in 4 5 minutes of scanning. If
the generic or specific determination of a diatom cannot
be made, it is recorded as unknown. When the count is
completed, the tallies are totaled, and the percentages of
the four most abundant species are calculated and recorded.
If the plankton counts are less than 500 per ml, the
centrifugation method may not provide enough diatom material
to prepare a countable slide. In this case the diatoms may
be concentrated from a larger volume of sample (l liter) by
allowing them to settle out. However, caution must be
exercised in the use of this method because it does not
quantitatively remove diatom cells smaller than 10|i in
diameter in less than Ik- days' settling; consequently, this
method can only be used safely and economically for samples
with large forms of diatoms.
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-11-
Figure lj. Settling Tube.
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Gravity drainage
to this level
Level I
Level II
Level III
Level IV
Figure 6. Lower Portion of a Settling Tube.
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In the settling method, the sample is thoroughly mixed
and approximately 1 liter is poured into a cylinder (Figure
5). After kQ hours the cylinder is emptied through a side
port, the drain valve and stopper are removed, and the vater
is lowered to level I (Figure 6) by use of a small suction
tube introduced through the drain port. The cylinder is
then swirled to loosen the deposits on the shoulder at the
lower end and allowed to stand 1 hour to permit the plankton
to resettle. The water is then lowered to level II, and the
cylinder is again swirled and allowed to stand 1 hour. The
process is repeated until the sediment has been deposited
in the vial. The vial is then removed, and a diatom slide
is prepared as described above.
E. Zooplankton Analysis
Rotifers and micro-crustacea are quantitatively removed
from the samples by settling 1 liter of sample 24 hours in
the cylinder as described in the preceding paragraph. If
more than a half inch of sediment collects in the vial, it
may be necessary to dilute the concentrate before the counts
can be made. The turbidity in sample vials containing lesser
amounts of solids can be removed by using the following method;
a. After standing 15 minutes, three-quarters of the
water above the sediment is withdrawn with a suction
tube.
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-1k-
b. The vial is refilled with tap water, inverted
several times, and allowed to settle 15 minutes.
c. Steps a and b are repeated as many times as
necessary to obtain a countable sample.
The zooplankton concentrate is then brought to a volume
of 8 ml, mixed well, and the entire sample is placed in a
counting chamber 80 X 50 X 2 mm (Figure 7), using the same
technique described for filling a Sedgwick-Rafter cell.
Figure 7. Zooplankton Counting Chamber.
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Rotifers
Ten strips across the chamber are scanned at 100X
(a fifth of the chamber), and the rotifers are identified
to genus. If no rotifers are encountered in the strips,
a zero count is recorded. If a tally of 100 is reached
for any genus before the count is nine-tenths complete,
the tally of that genus is discontinued at the end of the
strip "being counted, and that count is multiplied by a
factor to convert it to organisms per liter.
Crustacea
Nauplii are enumerated at the time of the rotifer count.
Adult copepods, cladocera, and other large forms are enumer-
ated under a binocular dissecting microscope at 20X by scanning
the entire contents of the zooplankton cell. Crustacea are
identified to genus only.
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-1.6-
II. Periphyton
A. Collection
The sampler consists of a styrofoam float approximately
12 X 12 X 2 inches, which supports a central plexiglass cradle
holding 1- X 3-inch glass microscope slides (Figure 8).
Generally, two slides are exposed at each station for 2 weeks.
However, the exposure time may vary, depending upon arrange-
ments made with local cooperating personnel. At the end of
the exposure period, the slides are removed from the sampler,
placed in a 3-ounce "bottle containing approximately 70 ml of
yjo formalin, and shipped to our laboratory. A bottle contain-
ing preservative, a sample data tag (see Appendix), and clean
slides are mailed to the station in advance of the collection
of the sample (Figure 9)« The mailing container is supplied
with a franked, return address label.
B. Preservation
A 5$ formalin solution is prepared by diluting technical
grade formaldehyde solution (37% HCHD) with distilled water.
C. Sacrple Preparation
With a razor blade, the periphyton is scraped from the
slides into the 3-ounce sample bottle, and preservative is
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Figure 8. Beriphyton Sampler.
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added to bring the total volume to 90 ml. At this time, 5 to
8 ml of the sample is poured into n disposable 3-dram vial and
set aside for diatom slide preparation.
v_
Figure 9- Periphyton Sample Bottle.
D. Sedgwick-Rafter Cell Analysis
After thoroughly mixing the sample by repeatedly filling
and discharging a straight-sided pipette (inside diatmeter
3 mm) in the bottle, 1 ml is transferred to a Sedgwick-Rafter
cell, and a strip count is made. The counting procedure is
same as that outlined in the plankton section, except that a
cell count is made of all organisms (see bench sheet in Appendix).
If the organisms are too concentrated to permit a direct count,
a 1-ral aliquot is diluted to 5 ml, and the material is placed in
the Sedgwick-Rafter cell. Further dilution is occasionally
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necessary. The scrapings may contain clumps of cells, even
after the sample is thoroughly shaken. This may result in
a more uneven distribution of material in the counting cell
than occurs vith the plankton samples, but it cannot be
entirely avoided.
E. Diatom Species Proportional Analysis
The same procedures (and bench sheet) used for the
preparation and counting of plankton diatoms are used to
process the periphyton samples, except that a chemical
treatment is frequently used to separate the aggregates
(colonies) of diatoms into individual cells. In this case
the intercellular gelatinous matrix is digested with the
oxidant, potassium persulfate (KpSpOo). Prior to the
oxidation step, the formalin solution is decanted from the
diatom sample vial vith a suction tube. A 5$ KpSpOn solution
Q
is added, and the sample is heated to 95 C for at least 30
minutes. The sample is then allowed to cool and settle for
2k hours. The KpSpOn solution is decanted with a suction
tube, and the vial is refilled with distilled water and
allowed to stand 2k hours. A minimum of three changes of
distilled water are necessary to remove enough of the residual
salt from the sample so that a crystalline layer does not form
when the material is dried on the coverglass.
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Appendix
1. Plankton Tag and Sampling Reminder.
2. Periphyton Tag.
3. SedgvrLck-Rafter Plankton Bench Sheet.
k. Diatom Bench Sheet.
5. Periphyton Sedgwlck-Rafter Bench Sheet,
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WATER POLLUTION SURVEILLANCE SYSTEM
1014 Broadway, Cincinnati, Ohio ^5202
FLANKTOH SAMPLE
River_
Station
Date ___
CollectecTby
NOTICE
Whenever possible, plankton samples should be
collected during the first full week of each
month. This sample bottle should be filled
and shipped during the week of
Extra bottles that accumulate because of missed
samples should be returned empty to the Water
Pollution Surveillance System (formerly the
National Water Quality Network) in Cincinnati.
WATER POLLUTION SURVEILLANCE SYSTEM
1014 Broadway, Cincinnati, Ohio 45202
PERIPHYTON SAMPLE
River. _
Station „
Date ln_ Date Out
Collected By
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River o'r Lake
Station
State
Pennates
First check_
Recorded
NL-C-1A
(5-66)
SEDGWICK-RAFTER DATA
Date Analyzed
Analyzed by
Station No.
Date
Collected
CODE
ORGANISM
TALLY
C/ML.
Total coccoid blue-green algae per ml. -f.
Total filamentous blue-green algae
TOTALS
/
Total coccoid green algae-
Total filamentc
Total eon
Total other pigmen
IUB gre<
5en flaj
tedfjUj
:n algae <
jellate s-^
jellates -<
\
\
\
/
\
Gentries c/ml.
Most
Diatoms
c/ml.
Abui
Alg
idant Centric
le Shells
Live
Pennate
Meloa . | Others Totals
c/ml
Total live centric diatoms -<
c/ml.
Shells
Live Pennates
S-R
/
Total live pennate diatoms-^
TOTAL LIVE ALGAE
Remarks:
Wash, sheet
Wash, sheet checked
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ANIMAL ANALYSTS
COOS
11
02
17
21
22
OROAHIBM
BOTIFERA
Kerat«lla
Braehioaus
Polyarthra
Synchaeta
Trichocera
TALLY
C/LITER
Total Rotifers per llter-f
51
S2
53
CLADOCXRA
Jocmln*
ITaphn<«
MoiJU,
Ceriodaptmia
COPBPODA
50
76
77
Nauplll
Cyclops &
related genera
Dlaptaua
Total Crustacea per lit«r<
NEMATODE3(per liter)
OTHER INVERTEBRATES: (per liter)
(
\
Most
Abundant
Rotifers
Most
Abundant
Crustacea
Factor
Analyzed by
Date Analyzed
Code to
Species
Percentage
Diatom Percent Abundance
(Fran diatom bench sheet)
1st
2nd
3rd
^th
1.-C-14
{ >-66)
Percent others
Total #of species
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River
Live Gentries
Live Pennates
Total Live
S-R Count
DIATOM ANALYSIS
Station State
Dead Gentries
Dead Pennates
Total Dead
Station Number
Date Collected
Analyzed by
Date Analyzed
Counting Time
Species
Coscinodiscus
\
Cyclotella
Meaeghiniana
Melosira
Ambigua
granulata
distans
f Rhizosolenia
" Stephanodiseus
hantzschii
invisitatufl
astrea minutula
(Other Gentries
I
fAchnanthes
Amphiprora
Aaphora
f Asterionella formosa
f Caloneis
[ Cocconeis
Cymatopleura
/ Cymbella
Diatooa vulgpxe
' Dinloneis smithii
Epithemia
Eunotia
Total
FIRST SECOND THIRD FOURTH
%
Percent
Code others
*
No. species
Species
"Fragilaria crotonensls
conatruens
f Frostulia
Gomphonema
? Gomphoneis
Gyros igma
(Meridion eirculare
Navicula
pitzschia
f Pinnularia
I
Pleurosigma
Rhoicosphenia curvata
rstauroneis
(Rhopalodia
rsurirella
Synedra
ulna
acus
Tabellaria
fenestrata
flocculosa
Total
f-
DQ™=-rVB- Total coum;
NL-C-15
(6-66)
-------
(9-65)
River or Lake
Station
State
Periphyton Data
Inclusive Dates
Date Analyzed
Analyzed by
CODE ORGANISM
Tuny
c/ml
e/am^ e/mm^
j
/
Total Filamentous blue-/treen algae
Total coceoid green algae
Total Filamentous green algae
Total green flagellates
Other cocco
id algae
Other pigmented flagellates
Gentries
c/ml
Penmates
c/ml
Most
abundant
Diatoms
c/ml
Centric shells
Live Gentries
c/mm
Total live centric diatoms
Pennate shells
Live pennates
Total live pennate diatoms
S-R Factor
Preservative
No. slides collected_
Area scraped
REMARKS:
TOTAL LIVE ALGAE
(cells/ran2)
Scrapings diluted to
ml
First check_
Recorded
Wash, sheet
Wash, sheet checked
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