c/EPA
United States
Environmental Protection
Agency
Office of
Research and
Development
Environmental Research
Laboratory
Corvallis, Oregon 97330
EPA-600/9-78-018
July 1978
THE SELENASTRUM
CAPRICORNUTUM
PRINTZ ALGAL ASSAY
BOTTLE TEST
Experimental Design,
Application, and Data
Interpretation Protocol
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EPA-600/9-78-018
July 1978
THE
SELENASTRUM CAPRICORNUTUM PRINTZ
ALGAL ASSAY BOTTLE TEST
Experimental Design, Application,
.and Data Interpretation Protocol
by
William E. Miller, Joseph C. Greene
and Tamotsu Shiroyama
Special Studies Branch
Corvallis Environmental Research Laboratory
Con/all is, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Pro-
tection Agency would be virtually impossible without sound scientific data on
pollutants and their impact on environmental stability and human health.
Responsibility for building this data base has been assigned to EPA's Office
of Research and Development and its 15 major field installations, one of
which is the Corvallis Environmental Research Laboratory (CERL),
The primary mission of the Corvallis Laboratory is research on the
effects of environmental pollutants on terrestrial, freshwater, and marine
ecosystems; the behavior, effects and control of pollutants in lake systems;
and the development of predictive models on the movement of pollutants in the
biosphere.
This report reflects the latest research findings of the continued
refinement, evaluation and application of algal assays to study the effects of
pollutants upon algal productivity in natural waters. This test protocol can
be used to evaluate nutrients, heavy metals, new product formulations and
complex wastes.
A. F- Bartsch
Director, CERL
m
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PREFACE
This document is the product of intensive research to improve and expand
the understanding of results obtained from the Algal Assay Procedure: Bottle
Test (USEPA, 1971) to enable investigators to define the stimulatory and/or
inhibitory interaction(s) of municipal, industrial and agricultural wastes
upon algal productivity in natural waters.
This research was designed to determine:
(1) The impact of nutrients and/or changes in their loading upon algal
productivity;
(2) Whether the growth response of Selenastrum capricornutum reflects
the response of indigenous species;
(3) The feasibility of the assay test protocol to evaluate heavy metals;
(4) The capability of the assay to define the effect(s) of complex
wastes; and
(5) If the assay information can be applied to define and assist in the
management of real-world situation.
As a result of these research efforts the Selenastrum capricornutum
Printz Algal Assay Bottle Test: Experimental Design, Application and Data
Interpretation Guide is offered now for wider application in both eutrophi-
cation and toxicity problem areas. This point in progress has been attained
through the dedication and continuing energies of Mr. Miller, Mr. Greene and
Mr. Shiroyama. To them goes much credit for the effective way in which the
research effort moved continuously and effectively toward the refinement and
application of the Algal Assay Procedure: Bottle Test.
The research could not have been completed without the efforts of Ethan
Bergman, Kurt Putnam, Ellen Merwin, Mike Long and Amy Leischman and others who
provided laboratory support on various research projects.
Special appreciation is also extended to Amy Leischman and Mike Long for
editing the bibliography and compiling the mailing list. The untiring,
cheerful support of Nancy Cruse, who typed this document and suffered through
its many revisions is also greatly appreciated. Acknowledgement is also given
to Howard Mercier for providing the FORTRAN data reduction program.
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TABLE OF CONTENTS
Page
FOREWORD. i-ji
PREFACE -jv
INTRODUCTION 1
PRINCIPLE 4
PLANNING OF ALGAL ASSAYS 8
Selection of Test Waters 8
Sample Collection, Transport, Preparation and Storage 9
APPARATUS 11
Sampling and Sample Preparation 11
Culturing and Incubation 11
Bioassessment Evaluation 12
SYNTHETIC ALGAL NUTRIENT MEDIUM . M
Final Concentration of Nutrients 18
Storage of Culture Medium 19
TEST ALGA 19
Source of Test Alga 20
Maintenance of Stock Culture 20
Preparation of Inoculum 21
TEST CONDITIONS 22
Temperature 22
Illumination 22
Gas Exchange 22
PROCEDURE 22
Preparation of Glassware 22
pH Control 23
Growth Parameter 24
Laboratory Measurement 25
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TABLE OF CONTENTS (Cont'd)
DATA ANALYSIS 28
Confidence Intervals 28
Rejection of Outliers 36
EXPERIMENTAL DESIGN AND ANALYSIS 38
Nutrient Limitation 38
Phosphorus Limitation 40
Nitrogen Limitation 44
Trace-element Limitation 48
Co-1 imitation 50
Application of Nutrient Limitation Studies 50
Heavy Metal Toxicity 53
Experimental Design 54
Interpretation of Results ..... 55
New Product Evaluation 57
Introduction 57
Experimental Design 57
Evaluation of Complex Wastes 61
LITERATURE CITED. ...... 65
APPENDICES 68
Selenastrum sp. Bibliography 68
Recommended Equipment and Supplies 80
FORTRAN Data Reduction Program 83
Standard Algal Assay Forms ..... . 99
Algal Growth Potential 101
Dilution Test 103
Dose Response Test 105
Growth Assessment Cover Sheet 107
Growth Assessment Data (short form) 109
Growth Assessment Data (long form) .111
Computer ID Form 113
Dry Weight versus Days. 115
Dry Weight versus Any x Value 117
Cell Counts versus Days 119
Dry Weight versus Cell Counts 121
Mean Cell Volume Calibration Procedure 122
VI
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1.0 Introduction
Algae are natural inhabitants of waters and are an extremely important
group of plant organisms. Through their photosynthetic activity they help to
provide the oxygen necessary for the survival of animal species found in the
aquatic environment. Algae contribute to the self purification of streams,
lakes and estuaries, and also serve as the basis of the food chain within the
aquatic ecosystem. However, when nutrients and sunlight are plentiful they
are capable of rapid growth and multiplication. This often results in serious
water quality problems. In the absence of sunlight they deplete the oxygen
levels through their respiratory activity. Heavy growths, or "algal blooms"
often cause tastes and odors in drinking water supplies. Some algal species
produce metabolic products that are toxic and have been implicated in the
death of livestock, waterfowl and fish. Because of the widespread interest in
algae, strong emphasis has been placed upon having a standard and reproducible
method for determining the potential of waters, sewage and industrial efflu-
ents, and various compounds to support, accelerate or inhibit algal growth.
The significance of measuring the algal growth potential of water is that
a differentiation can be made between the nutrients that are in the sample (as
determined by chemical analysis) and the nutrient forms that are actually
available for algal growth. The addition of a given nutrient(s) to a sample
can give an indication of which nutrient(s) is limiting for algal growth.
Also, if algal growth remains limited when nutrients are in sufficient supply
and the physical conditions for growth exist, the presence of a toxicant is
indicated (Miller, Maloney and Greene, 1974; Greene et a!., 1975; Payne, 1976;
Gerhold, 1976; Greene et al_. , 1976).
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The interpretation of actual algal assay results depends on the relia-
bility of the test procedure. To be effective an assay experiment should be
designed to include built-in checks and balances of known growth responses,
such as to standard additions of nitrogen and phosphorus singularly or in com-
bination. The amount of growth response of the test organism can be used to
verify both chemical analyses for nitrogen and phosphorus and the precision
and accuracy of the assay response. Failure of a test water to attain the
predicted yield or nutrient limitation status can usually be attributed to one
or more of the following causes: (1) absence of other growth requiring nu-
trients; (2) the presence of toxicants; or (3) unreliable chemical analysis
for Ortho-P and total soluble inorganic nitrogen (N02 + N03 + NH3-N = TSIN).
The use of standard laboratory algal test species, grown under specific
environmental culture conditions, is essential to the understanding of the
complex interaction of nutrient and/or inhibitor laden wastes upon aquatic
productivity. Odum (1971) discussed the use of unialgal cultures as being
prerequisite to defining the growth effect of each nutrient in relationship to
the combined effects of all other factors within the entire complex of con-
ditions. Detection of algal growth reactions, whether inhibitory or stimu-
latory, becomes more precise as detailed background information accumulates on
the physiology of a given test species.
When comparing algal growth potentials from a number of widely different
water sources there are advantages in using the same species of algae for all
waters. The alga to be used must be readily available and its growth must be
able to be measured easily and accurately. It must also respond to growth
substances uniformly. Some algae are capable of concentrating certain nu-
trients in excess of their normal metabolic requirements. Therefore, this
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factor must be taken into account in selecting the culture medium and in
determining the type and amount of algae to use. If algae are cultured in a
relatively dilute medium, as recommended in the "Algal Assay Procedure:
Bottle Test" (USEPA, 1971), the amount of growth in subsequent testwaters
resulting from nutrient carryover is minimized. Experiments with the green
alga, Chiorella pyrenoidosa, grown in this relatively dilute medium, dis-
closed no significant further growth in media lacking nitrogen or phosphorus.
This was true even when these algae were transferred from the initial medium
over a wide range of inoculum sizes (Fitzgerald, 1972).
Isolation of a single indigenous algal species, even if that alga were
dominant at the time of sampling, does not mean that when grown in laboratory
culture it is more indicative of natural conditions than a laboratory species.
The use of an indigenous algal species isolated for use as a specific labora-
tory test organism is not recommended. The dynamics of natural phytoplankton
blooms, in which the dominant algal species changes throughout the growth
season, makes it quite certain that even if the indigenous algal isolate were
dominant at the time of collection, many other species will dominate the
standing crop as the season progresses.
The presence of indigenous algae in a water sample suggests that they are
the most fit to survive in the environment from which the sample was taken.
Under adequate light and temperature conditions the indigenous algae should
produce biomass until growth is limited by some essential nutrient or inhib-
itor. If the indigenous algae are limited from further growth by an essential
nutrient, the laboratory test alga cultured in a non-competitive environment
and responding to the same limiting nutrient will produce parallel maximum
growth yield responses.
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Generally, indigenous phytoplankton bioassays are not necessary unless
there is strong evidence of the presence of persistent sub-lethal toxicants to
which indigenous populations might have developed tolerance (Greene et al. ,
1978).
The extensive design, evaluation and application of algal assay research,
centered around the use of Selenastrum capricornutum as the dominant test
alga, has demonstrated the ability of unialgal assays to identify and assist
in the management of major water quality problem areas. This document is the
result of extensive research using the "Algal Assay Procedure: Bottle test,"
developed by the Environmental Protection Agency, for assaying algal growth
potential in natural water samples (USEPA, 1971). It is this work on which
the following test is based.
2.0 PRINCIPLE
This assay procedure is based upon a modification £f Liebig's Law erf the
minimum which states that "maximum yield is proportional to the amount of a
nutrient or combination of nutrients which are present and biologically avail-
able in minimal quantity in respect to the growth requirements of the organ-
isms." As stated by Liebig, his law applies to a single nutrient limiting
growth at any one time. This concept has been documented for the critical
nitrogen and phosphorus requirements for optimum growth of ^. capricornutum in
both culture medium and natural waters providing other essential elements are
present JHI excess (Shiroyama, Miller and Greene, 1975). However, the concept
of a single limiting nutrient is not infallible. More than one nutrient can
simultaneously limit growth. For example: the interaction of nitrogen and
phosphorus can regulate maximum yield of S. capricornutum as the critical
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ratio of these elements approaches 11:1. Algal growth can often be stimulated
in test waters containing this ratio of N:P by the combined addition of N and
P spikes. These growth responses support the current modification of Liebig's
Law which is considered valid for the interpretation of nutrient limitation
obtained under conditions specified in this document.
The test in its present form is intended primarily for use in the fol-
lowing general situations:
1. Assessment of a receiving water to determine its nutrient status and
sensitivity to changes in N and P loading.
2. Evaluation of materials and products to determine their potential
stimulatory or inhibitory effects on algal growth in receiving
waters.
3. Assessment of effects of complex wastes originating from industrial,
municipal, and agricultural point or non-point sources to define
their impact upon receiving waters.
The bottle test consists of three steps: (1) selection and measurement
of biomass parameters during the assay (for example, biomass indicators such
as dry weight); (2) presentation and statistical evaluation of the measure-
ments made during the assay; and (3) interpretation of the results with re-
spect to the specific problem being investigated. It is intended that the
test be used: (1) to identify algal growth-limiting constituents; (2) to
determine biologically the availability of algal growth-limiting nutrients;
and (3) to quantify the biological response to changes in concentrations of
algal growth-limiting constituents. These measurements are made by adding a
selected test alga to the test water and determining algal growth (as dry
weight) at appropriate intervals.
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The test also may be used to determine whether or not complex wastes,
inorganic or organic compounds, or receiving waters are inhibitory to algae.
Caution should be observed in interpreting results where there is little or no
growth response in samples when sufficient nutrients appear to be or are, in
fact, present. The presence of toxicants can inhibit or prevent algal growth
even when nutrients are not growth-limiting.
It should be pointed out that test flasks are normally incubated to
facilitate free gas exchange at the air-water interface. Therefore, carbon
dioxide is rarely growth-1imiting except in cases where maximum yield exceeds
200 mg dry weight I-1. Because of this design feature, the test as outlined
cannot be used to define growth limitations of carbon in the test water. The
test can be modified to obtain such information.
2.1 Growth response—Maximum standing crop (MSC) is proportional to the
initial amount of limiting nutrient available providing other factors are not
growth regulating. All comparative growth responses should be analyzed sta-
tistically and significant levels of the differences should be reported.
A statistical coefficient of variance analysis of the MSC replication
obtained in 685 test waters (each consisting of 3 replicate flasks) for yields
ranging between 0.01 and 130.00 mg dry wt I-1 are shown in Table 1.
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TABLE 1
STATISTICAL COEFFICIENT OF VARIANCE ANALYSIS OF THE
STANDING CROP (MSC) REPLICATION
No Samples MSC % Coefficient of Variance
66
80
26
40
.27
27
29
31
22
25
14
86
39
20
11
2
7
7
13
33
32
11
17
14
2
4
0.01 -
0.10 -
1.00 -
2.00 -
3.00 -
4.00 -
5.00 -
6.00 -
7.00 -
8.00 -
9.00 -
10.00 -
15.00 -
20.00 -
25.00 -
30.00 -
35.00 -
40.00 -
50.00 -
60.00 -
70.00 -
80.00 -
90.00 -
100.00 -
110.00 -
120.00 -
0.09
0.99
1.99
2.99
3.99
4.99
5.99
6.99
7.99
8.99
9.99
14.99
19.99
24.99
29.99
34.99
39.99
49.99
59.99
69.99
79.99
89.99
99.99
109.99
119.99
130.00
47.8
45,4
27.1
26.4
19.6
17.7
17.8
14.4
12.5
13.8
12.5
11.8
11.6
9.0
10.2
3.2
8.5
7.2
7.5
6.3
6.8
8.2
8.1
8.7
9.0
7.8
The coefficient of variance decreases as the MSC increases. The higher
values corresponding to MSC < 1.00 mg dry wt I-1 and the lower percent vari-
ance for yields > 10.00 mg dry wt I-1. The following percent variance guide-
lines can be used to ascertain whether the differences obtained in MSC between
replicate flasks and/or nutrient additions are statistically significant:
± 50% for MSC < 1.00 mg dry wt I-1
± 30% for MSC > 1.00 but < 3.00 mg dry wt I-1
± 20% for MSC > 3.00 but < 10.00 mg dry wt I-1
± 10% for MSC > 10.00 mg dry wt I-1
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3.0 PLANNING OF ALGAL ASSAYS
The specific experimental design of each algal assay is dictated by the
actual situation. It is extremely important that all pertinent environmental
factors be considered in the planning of a given assay to insure that valid
results and conclusions are obtained.
Resource availability (manpower, equipment, and dollars) often dictate
the degree of sophistication of the assay. Therefore, the following specifics
must be considered as an absolute minimum by each investigator who plans to
conduct algal assays for the purposes listed above (see 2.0).
3.1 Selection of test waters—Water quality may vary greatly with time
and with location in lakes, impoundments and streams. Sampling programs must
be established so that meaningful data will be obtained.
3.11 Spatial variations—In a thermally stratified lake or impound-
ment, only depth integrated euphotic zone composite samples need be collected.
In most cases, the euphotic zone is described as the depth to which at least
1% of the surface light is available. Euphotic depths greater than 8 meters
should be subsampled at least at the surface and at each 3-meter depth in-
terval. Likewise, euphotic zones less than 8 meters should be sampled at
least at the surface and 2-meter intervals. Each equal volume depth sample
must be composited in a suitable nonmetallic container and upon thorough
mixing is subsampled for algal assay and chemical and biological analysis--
including algal identification.
The use of transect lines are helpful in sampling. Samples from a tran-
sect can be taken from predetermined euphotic zones. Representative river
samples can be identified by specific conductance measurements which show the
homogeneity of the sampling transect. In rivers and streams useful infor-
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(nation may be obtained by taking samples upstream and downstream from sus-
pected pollutant sources or confluent tributaries.
New products should be evaluated for their stimulatory and/or inhibitory
effect upon algal growth before being discharged into receiving waters. When
new products or materials are evaluated, samples of natural waters from
geographically different areas having a range of representative water quality
(such as alkalinity, hardness, pH, and ionic strength) must be investigated.
3.12 Temporal variations—The nutrient content of natural and waste
waters often varies greatly with time. The variation may not only be sea-
sonal, but hourly. The effects of these variations in lakes and in impound-
ments must be considered and can be minimized when sampled in accordance with
section 3.11.
3.2 Sample collection, transport, preparation and storage.
3.21 Collection—Use non-metallic water sampler and autoclavable
storage containers (such as linear polyethylene, polypropylene, or polycar-
bonate). Containers should not be re-used when toxic or nutrient contami-
nation is suspected.
3.22 Transport conditions—Leave a minimum of air space in the
sample container, keep in the dark and packed in ice. (Taping the bottle cap
helps to insure against leakage.)
3.23 Preparation—In order to use a unialgal test species the
indigenous algae in the sample must be "removed" before assaying. This re-
moval requires destruction and separation of the indigenous algae. Auto-
el aving followed by filtration is recommended when it is desired to determine
the amount of algal biomass that can be grown from all nutrients in the water,
including those contained in filterable organisms and other particulate mat-
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ter, which can be solubilized by autoclaving. The sample should be autoclaved
at 1.1 kg cm2 (15 psi) at 121°C (250°F). The period of autoclaving will
depend on the sample volume, e.g., 30 minutes or 10 minutes per liter, which-
i
ever is longer. After autoclaving and cooling, the sample should be equil-
ibrated by bubbling with a 1% carbon dioxide and air mixture to restore the
carbon dioxide lost during autoclaving and to lower the pH to its original
level (it will generally rise on autoclaving). In some instances, waters with
total hardness greater than 150 mg I-1 will lose calcium and phosphorus upon
autoclaving. This precipitate may be resistant to resolubilization by addi-
tion of carbon dioxide and air. In waters containing high levels of hardness
and alkalinity the pH may not increase upon autoclaving. It is recommended
that 1% C02 and air mixture be bubbled through the sample for at least 2
minutes per liter. If an electronic particle counter is to be used for all
counting, the carbon dioxide equilibrated sample must be passed through a
0.45pm membrane filter.
Autoclaving followed by filtration is the recommended pretreatment for
nutrient limitation and heavy metal toxicity studies; however, its use in
studies of complex wastes and organic compounds may alter the chemical struc-
ture and bias the assay response. Presently, filtration (0.45 urn) is the only
recommended pretreatment prior to the assay of organic compounds and complex
wastes. Filtration is essential to eliminate unwanted biological contaminants
which would invalidate the growth response of the test organism.
3.24 Storage—Although changes can occur in pretreated water samples
during storage, regardless of storage conditions, the extent or chemistry of
these changes is not well defined. Attempts should be made to minimize the
10
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effect of storage by keeping samples cooled at 4°C in the dark, using proper
containers, and avoiding air spaces over the sample.
4.0 APPARATUS
4.1 Sampling and sample preparation.
4.11 Water sampler—Non-metallic
4.12 Sample bottles--Autoclavable (such as polypropylene, linear
polyethylene or polycarbonate).
4.13 Membrane filter apparatus—For use with 47 or 142 mm filter
pads and 0.45|jm porosity filters. To reduce filtration time, the larger
membrane (142mm) filtration unit is recommended.
4.14 Autoclave or pressure cooker—Capable of producing 1.1 kg cm2
(15 psi) at 121°C (250° F).
4.2 Culturing and incubation.
4.21 Culture vessels—Erlenmeyer flasks of good quality borosili-
cate glass such as Pyrex or Kimax. When trace nutrients are being studied,
special glassware such as Vycor, polycarbonate, or coated glassware can be
used.
The flask size is not critical but, due to carbon dioxide limitation, the
sample to volume ratios are. The recommended sample to volume ratios are:
25 ml sample in 125 ml flask
50 ml sample in 250 ml flask
100 ml sample in 500 ml flask
These twenty percent sample to volume ratios are for flasks which are
shaken by hand once daily. Maximum permissible sample to volume ratios in
continuously shaken (100 rpm) flasks should not exceed 50%.
11
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4.22 Shaker table—Capable of 100 revolutions per minute (Figures 1
and la). A schematic of the 500 ml flask platform is shown in appendix 11.2.
4.23 Culture closures—Foam plugs must be used to permit good gas
exchange and prevent contamination. Each laboratory must determine for each
batch of closures purchased whether that batch has any significant effect on
the maximum standing crop.
4.24 Constant temperature room or equivalent incubator—Capable of
providing temperature control at 24 ± 2°C (Figure 2).
4.25 Illumination--"Cool-White" fluorescent lighting to provide
4304 lumens (400 ± 10% ft-c) measured adjacent to the flask at the liquid
level (Figure 3).
4.26 Light metei—Several types are acceptable, but the meter must
be calibrated against a standard light source or light meter. Commercial
laboratories can perform such standardizations by comparison with calibrated
lamps supplied by the National Bureau of Standards. Standardization should
include both intensity and color temperature comparisons if the light meter is
designed for a color temperature different from the color-correlated tem-
perature (4200°K) of the "Cool-White" fluorescent light source specified in
the assay procedure.
4.27 pH meter—Scale of 0-14 pH units with accuracy of ± 0.1 pH unit.
4.3 Bioassessment evaluation
4.31 Electronic particle counter with mean cell volume computer (MCV).
4.32 Fluorometer—Suitable for measurement of chlorophyll a (see 8.53)
4.33 Microscope—General purpose.
4.34 Microscope i11uminator—Good quality general purpose.
12
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co
Figure 1. Shaker platform with 500 ml Erlenmeyer flasks,
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Figure la. Shaker platform with 125 ml Erlenmeyer flasks,
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t
c_n
Figure 2. Constant Temperature room.
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•.
~
Figure 3. Lightbank and support frame.
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4.35 Hemacytometer counting chamber and occular micrometer (used to
measure diameter of MCV reference standard).
The above equipment is listed in the order of use preference for moni-
toring biomass change.
4.36 Oven--Dry heat capable of temperature of 120°C.
4.37 Centrifuge—Capable of relative centrifugal force of at least
1,000 x g.
5.0 SYNTHETIC ALGAL NUTRIENT MEDIUM
Culture medium is prepared as follows: add one ml of each stock solution
in 5.1 through 5.7 in the order given to approximately 900 ml of distilled or
de-ionized water and then dilute to one liter. Adjust final medium pH to 7.5
± 0.1 with 0.1 normal sodium hydroxide or hydrochloric acid as appropriate.
Immediately filter the pH adjusted medium through a 0.45 urn membrane at a
vacuum not to exceed 380 mm (15 inches) mercury or at a pressure not to exceed
1/2 atmosphere (8 psi).
5.1 Sodium Nitrate Stock Solution: Dissolve 12.750 g NaN03 in 500 ml
distil led water.
5.2 Magnesium Chloride Stock Solution: Dissolve 6.082 g MgCl2-6H20 in
500 ml distilled water.
5.3 Calcium Chloride Stock Solution: Dissolve 2.205 g CaCl2-2H20 in
500 ml distilled water.
17
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5.4 Micronutrient Stock Solution: Dissolve in 500 ml distilled water:
92.760 mg H3B03 0.714 mg CoCl2-6H20
207.690 mg MnCl2-4H20 3.630 mg Na2Mo04-2H20
1.635 mg ZnCl2 0.006 mg CuCl2-2H20
79.880 mg FeCl3-6H20
150.000 mg Na2EDTA-2H20 [Disodium (Ethylenedinitrilo) tetraacetate]
5.5 Magnesium Sulfate Stock Solution: Dissolve 7.350 g MgS04-7H20 in
500 ml distilled water.
5.6 Potassium Phosphate Stock Solution: Dissolve 0.522 g K2HP04 in 500
ml disti1 led water.
5.7 Sodium Bicarbonate Stock Solution: Dissolve 7.500 g NaHC03 in 500
ml distil led water.
If desired, reagent salts 5.1 through 5.4 can conveniently be combined
into one 500 ml stock solution.
5.71 Final concentration of macronutrients as salts and elemental
concentration (mg I-1) of distilled or de-ionized water.
compound
NaN03
MgCl2-6H20
CaCl2-2H20
MgS04-7H20
K2HP04
NaHC03
concentration (mg I-1)
25.500
12.164
4.410
14.700
1.044
15.000
element
N
Mg
Ca
S
P
Na
K
C
concentration (mg I-1)
4.200
2.904
1.202
1.911
0.186
11.001
0.469
2.143
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5.72 Final concentration of micronutrients as salts and elemental
concentration (ng I-1) in distilled or de- ionized water.
compound
H3B03
concentration (ug I-1)
185.520
415.610
3.271
1.428
0.012
7.260
160.000
300.000
element
MnCl2-4H20
ZnCl2
CoCl2-6H20
CuCl2-2H20
Na2Mo04-2H20
FeCl3-6H20
Na2EDTA-2H20
5.73 Storage of culture medium—Culture medium must be filter-
sterilized (Sec. 5.0) or autoclaved. It is also recommended that uninoculated
sterile medium be stored in the dark at 4°C to avoid any (unknown) photochem-
ical changes.
B
Mn
Zn
Co
Cu
Mo
Fe
concentration (|jg I-1)
32.460
115.374
1.570
0.354
0.004
2.878
33.051
6.0 TEST ALGA
The recommended test alga Selenastrum capricornutum Printz is a green
alga (chlorophyceae) of the order chlorococcales. This alga was isolated from
the River Nitelva, in the County of Akershus, Norway, by Olav M. Skulberg,
Norwegian Institute for Water Research, 1959. Many green algae such as Chlo-
rel la, Scenedesmus , and Ankistrodesmus occur in waters of the most diversified
composition. Selenastrum belongs to this group of ubiquitous algae which have
a wide tolerance towards environmental conditions (Rodhe, 1978). Selenastrum
capricornutum is characterized by its unicellular habit in which the cells are
19
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in a non-motile condition throughout their entire life cycle. These attri-
butes allow this alga to be enumerated by an electronic particle counter.
6.1 Source of test alga--Available from the Environmental Protection
Agency, Corvallis Environmental Research Laboratory, Special Studies Branch,
200 SW 35th Street, Corvallis, Oregon 97330.
This test alga is also available (ATCC 22662) from the American Type
Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852.
6.2 Maintenance of stock culture:
6.21 Medium—See section 5.0
6.22 Incubation conditions--24 ± 2°C.
Under continuous "Cool-White" fluorescent lighting at 4304 lumens (400
± 10% ft-c), shaken continuously at 100 rpm.
6.3 Culture transfei—Upon receipt of the algal culture, a portion
should be aseptically transferred to the algal culture medium as prepared in
section 5.0. The volume transferred is not critical (approximately 1.0 ml),
however, be sure enough cells are included to overcome significant growth lag.
(i.e., 1.0 ml of algal culture in 25 ml of medium in a 125 ml Erlenmeyer flask
if not continuously shaken or 1.0 ml culture added to 50 ml in 125 ml flask
when shaken continuously.) The rest of the culture can be maintained up to
six months in a dark refrigerator at 4°C.
6.4 Subsequent stock transfers—Weekly aseptic routine stock transfer is
recommended to maintain a continuous supply of "healthy" cells for experi-
mental work. Extreme care should be exercised to avoid contamination of stock
cultures. To retain a unialgal culture over a long period of time it is
advantageous to prepare a semi-solid medium containing 1.0% agar. This semi-
solid medium is placed in sterile Petri plates. A portion of a liquid algal
20
-------
culture is streaked onto it and incubated under conditions in 6.22. Algae
should be transferred onto fresh plates every four weeks. Fresh liquid cul-
tures should be started by transfer of a single algal colony to liquid medium
at four week intervals. For regular inoculation, liquid cultures are superior
since agar cultures usually are not uniform because the cell layers on the
agar surface are differentially supplied with light and nutrients (as a result
of shading and diffusion).
6.5 Preparation of inoculum—Rinse algal inoculum free of culture
medium as follows: Fill centrifuge tube with 7-10 day stock culture and
centrifuge at 1000 x g for 5 minutes. Decant the supernatant and resuspend
the cells in sterilized distilled water. Repeat the centrifugation and de-
cantation step and resuspend the cells in distilled water prior to determining
the initial cell concentration.
After determining the initial algal cell counts the following equation
can be used to prepare the inoculum:
(A) (B) (C)
FINAL CONCENTRATION
FINAL VOLUME x OF INOCULUM x VOLUME OF SOLUTION
OF INOCULUM IN TEST FLASK IN TEST FLASKS
(ml) (cells ml-1) (ml) = Q
(D)
INITIAL ALGAL CELL COUNTS (cells ml-1)
IN THE WASHED STOCK CULTURE
Example: 180 flasks containing 100 ml of solution (C) are required for the
test. Each flask is to be inoculated with 1000 cells ml-1 (B) final concen-
tration. 200 ml of suspended algal cells (A) should be prepared to insure an
adequate amount of inoculum. The product of A, B, and C is divided by the
initial algal cell count (D). The resulting quotient (Q) indicates the volume
21
-------
(ml) of the initial stock culture suspension (D) to be added to the volumetric
flask (A) before bringing the solution up to volume. This inoculum solution
should contain a final concentration of 100,000 ± 10% cells ml-1, one ml of
which (when added to 100 ml of test solution) results in a final algal cell
concentration in the test flask of 1000 cells ml-1.
7JD TEST CONDITIONS
7.1 Temperature--24 ± 2°C.
7.2 Illumination—Continuous "Cool-White" fluorescent lighting 4304
lumens (400 ± 10% ft-c).
7.3 Gas exchange—Free exchange through foam plugs, shaken at least once
daily (see sample to volume reference in section 4.21) or at the preferred
rate of 100 rpm.
8.0 PROCEDURE
8.1 Preparation of glassware—The recommended procedure is as follows:
All cylinders, flasks, bottles, centrifuge tubes and vials are washed with
detergent and rinsed thoroughly with tap water. This is followed by a rinse
with 10% solution (by volume) of reagent hydrochloric acid (HC1); vials and
centrifuge tubes are filled with the 10% HC1 solution and allowed to remain a
few minutes; all larger containers are filled to about one-tenth capacity with
HC1 solution and swirled so that the entire inner surface is bathed. After
the HC1 rinse, the glassware is neutralized with a saturated solution of
Na2C03, then rinsed five times with tap water followed by five rinses with de-
ionized or distilled water.
22
-------
Disposable pipettes may be used to eliminate the need for pipette washing
and to minimize the possibility of contamination.
Cleaned glassware is dried at 50°C in an oven and is then stored either
in closed cabinets or on open shelves with the tops covered with aluminum
foil.
The recommended procedure for culture flask preparation is as follows:
Brush the inside of flasks with a stiff bristle brush to loosen any attached
materials. Wash with non-phosphate detergent and rinse thoroughly with tap
water. Rinse with a 10% solution (by volume) of reagent grade hydrochloric
acid (HC1) by swirling the HC1 solution so that the entire surface is covered.
Neutralize with saturated sodium carbonate solution (Na2C03). The glassware
should be rinsed thoroughly with distilled water. If an electronic particle
counter is to be used, the final rinse must be with 0.22 micrometer membrane
filtered distilled water. Dry the flasks in an oven at 50°C. Insert foam
plugs and autoclave for 20 minutes at 1.1 kg cm2 and 121°C. The caoled flasks
can be stored in closed cabinets until needed.
8.2 pH Control—To insure the availability of carbon dioxide the pH
should be maintained below 8.5. This can be accomplished by (1) using optimum
sample to volume ratios; (2) continuously shaking the flask (approximately 100
revolutions per minute); (3) ventilation with air or air/carbon dioxide mix-
ture; and, in extreme cases, by (4) bubbling an air/carbon dioxide mixture
through the culture. The growth response of S. capricornutum cultured in
algal culture medium adjusted either with sodium hydroxide or hydrochloric
acid to obtain initial pH values ranging from 3.0 to 11.0 in single unit
increments, is shown in Table 2.
23
-------
TABLE 2
THE EFFECT OF INITIAL pH UPON THE GROWTH RESPONSE
OF S. capricornutum CULTURED IN ASSAY MEDIUM
Initial p_H Maximum yield mg dry wt I-1
3.0 0.20
4.0 0.33
5.0 79.69
6.0 89.30
7.0 87.95
8.0 90.02
9.0 82.32
10.0 101.22
11.0 75.10
The resultant growth suggests that initial pH values ranging between 6.0
and 10.0 have no adverse effect upon the 14-day maximum yield of the test
alga, when cultured under free gas exchange conditions.
8.3 Growth parameter—The parameter used to describe growth of the test
alga is maximum standing crop expressed as dry weight. The maximum standing
crop in any flask is defined as the maximum biomass achieved during incuba-
tion. For practical purposes, it may be assumed that the maximum standing
crop is obtained within 14 days or whenever the increase in biomass is less
than 5% per day.
Growth rate should not be used as a growth parameter in batch cultures
since growth rate is indirectly related to external nutrient concentrations.
This explains why phytoplankton in natural waters may grow at their maximal
rate even when there is not a significant amount of the limiting nutrient in
24
-------
the water. For the same reason, phytoplankton may also grow at different
rates even when exposed to the same external nutrient concentration. There-
fore, the conventional Monod equation, which predicts growth rate in terms of
external nutrient levels, does not adequately describe the growth of phyto-
plankton. Specific details and the scientific rationale concerning growth
rate interactions can be found in the following references: Thomas and Dod-
son, 1968; Golterman et aj. , 1969; Eppley and Thomas, 1969; Rhee, 1972; Swift
and Taylor, 1974.
8.4 Laboratory measurement—After the maximum standing crop has been
achieved, the dry weight of algal biomass may be calculated indirectly or
determined gravimetrically. If biomass is determined indirectly, the results
should be converted to an equivalent dry weight using appropriate conversion
factors. For example: Electronic particle counts and associated mean cell
volumes (MCV) of S. capricornutum can be converted to calculated dry weight in
mg I-1 by the following equation:
CELL COUNTS x MCV
(Cells ml-1) (Cubic x [3.6 x 10-7] mg dry weight
micrometers) = S. capricornutum I-1
Caution: This equation is valid only when the MCV computer has been cali-
brated with an appropriate reference particle, i.e. # 13020 60 urn3 standard
verified and supplied by Coulter Electronics Inc., Hialeah, Florida. A maxi-
mum of 199 urn3 can be read directly from the MCV computer. The MCV of S.
capricornutum can increase beyond 199 pm3 when cultured in test waters con-
taining heavy metals, pesticides and complex industrial wastes. Adjustment of
either the amplification or aperture current will electronically reduce or
increase the mean cell volume readout by a constant factor. This allows
25
-------
calibration or scale readouts for particles greater than 199 pm3. A change in
amplification setting from % to 1.0 results in a multiplication factor of 2.0
(1.0 -r Jj), i.e., a direct scale readout of 110 pm3 at an amplification setting
of 1.0 is actually 220 pm3 (110 x 2).
The MCV calibration for Coulter Counter models ZB, ZBI and ZF is pre-
sented in appendix 11.5.
8.5 Biomass monitoring-^Several methods may be used, but they must
always be related to dry weight. The following methods are listed in order of
preference.
8.51 Dry weight—Indirect electronic particle counting
The principle of operation is as follows: the S. capricornutum cells are
suspended in a 1% sodium chloride electrolyte in a ratio of 1.0 ml cell
suspension to 9 ml of 0.22 pm filtered saline (10:1 dilution). The resulting
suspension is passed through a 100 urn diameter aperture. Each cell that
passes through the aperture causes a voltage drop proportional to its dis-
placed electrolyte volume which is recorded as a count. The knowledge of both
the number of particles (cells) per unit volume of sample (usually 1/2 ml) and
the change in mean particle (cell) volume, allow changes in cell biomass (mg
dry wt I-1) to be calculated reproducibly and accurately, using the equation
as outlined in section 8.4.
8.52 Dry weight—Gravimetric
Method I--A suitable portion of algal suspension is centrifuged, the
sedimented cells washed three times in distilled water, transferred to tared
crucibles or aluminum cups, dried overnight in a hot air oven at 70-75°C and
weighed. This method is more sensitive than Method II, but is open to error
through loss of cells during washing.
26
-------
Method II--This method involves filtering a measured portion of algal
suspension through a tared Mi Hi pore® filter. The filter recommended is type
BD with an 0.60 micrometer pore size.
The method is as follows:
(1) Dry filters for two hours at 70°C in an oven. (Temperatures above
75°C will close the membrane pores).
(2) Cool filters in a desiccator containing desiccant for at least one
hour before weighing.
(3) Filter a suitable measured aliquot of the culture under a vacuum of
380 mm of mercury (or at a pressure not to exceed 1/2 atmosphere).
Normally 10 ml is sufficient, but in thin cultures more may be
required.
(4) Rinse the filter funnel with 50 ml distilled water using a wash
bottle and allow the rinsings to pass through the filter. This
serves to transfer all of the algae to the filter and washes the
nutrient salts through the filter.
(5) Dry the filter to constant weight at 70°C, cool in a desiccator for
one hour and weigh.
8.53 Chlorophyll a--In vivo fluorescence of algal chlorophyll has
been used with many types of algae and has proved particularly useful with ^.
capricornutum and with indigenous algae or filamentous forms not easily meas-
ured at low concentrations by direct microscopic, gravimetric dry weight and
absorbance methods. This method is sensitive and can be quickly performed.
However, chlorophyll to cell mass ratio may vary significantly with growth in
natural waters having different chemical composition (Kuhl and Lorenzen,
1964). Chlorophyll measurement is unsatisfactory to assess the toxic or
27
-------
stimulatory effects of complex wastes which may absorb and fluoresce in the
same spectral region. I_n vivo fluorescence measurements can aid in evaluating
increases in cell biomass attributed to increased growth in specific test
waters, but should not be used to predict universal chlorophyll a to dry
weight biomass relationships.
8.54 Direct microscopic enumeration--Hemacytometer
8.55 Absorbance--The use of turbidity for algal cell measurements
is strongly discouraged. Table 3 presents the relationship between the dif-
ferent biomass monitoring methods. Note that there was no definition between
5,000 and 115,000 cells ml-1 when assessed as absorbance utilizing a spectro-
photometer at 750 nm (cell path of 1 cm).
9.0 DATA ANALYSIS
9.1 Introduction—The fundamental measure used in this Algal Assay:
Bottle Test to describe algal growth is the maximum dry weight mg I-1 (stand-
ing crop) produced during the 14-day incubation period. Other biomass indi-
cators such as those listed in section 8.5 may be used; however, all results
presented must include experimentally determined conversion factors between
the indicator used and the dry weight of S. capricornutum obtained.
9.2 Confidence intervals—The maximum standing crop should be presented
with the confidence interval indicated. The calculation of confidence inter-
val for the average values presented must be based on at least three samples.
Consequently, a minimum of three replications per sample and/or sample treat-
ment must be analyzed when a source water is studied. The results of these
three replicates are then used to calculate the standard deviation. Confi-
dence intervals are based upon the standard deviation (a).
28
-------
TABLE 3
RESULTS OF BIOMASS ASSESSMENT TECHNIQUES PRODUCED IN ALGAL ASSAY PROCEDURE LABORATORY CLASSES
CELL COUNTS (Cells ml"1)
ELECTRONIC
a k
ORIGINAL3
1
1
1
1
1
a
,949,125
,320,000
,320,000
,023,815
,023,815
501,737
501 ,737
458,000
458,000
115,226
115,226
57,000
50,031
50,031
10,340
10,473
10,473
5,846
5,846
Counts of..
STUDENT"
2,290,490
1,239,578
1,698,888
1,159,050
887 ,373
573,902
513,434
533,500
521 ,256
108,580
117,331
58,647
48,978
52,010
9,070
10,868
13,208
5,791
5,543
DRYd
WEIGHT
HEMACYTOMETER1- mg
2,475
2,125
1,075
950
1,135
680
360
470
675
85
110
85
305
65
10
63
10
4
5
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,000
,500
,000
28
14
21
15
10
7
6
I''
.40
.50
.51
.00
.80
.50
.50
6.60
6
1
1
0
0
0
0
0
0
0
0
Selenastrum capricornutum produced on a Coul
STAFF. ''Electronic eel
cell countshX mean cell
1 counts taken
volume X 2.0 X
by students.
10"7. Optical
.46
.30
.50
.68
.60
.70
.10
.10
.20
.07
.07
ABSORBANCE6
1 cm cell path
9 750 Nm
0.060
0.056
0.060
0.043
0.045
0.021
0.024
0.015
0.019
0.005
0.005
0.005
0.005
0.005
0.000
0.000
0.000
0.000
0.000
FLUORESENCE
TURNERf TURNER9 PRODUCT IV ITYh
Mod 111
11,400
11,500
10,750
11,250
10,744
2,400
1,829
3,650
3,150
20
53
330
10
14
18
3
3
24
26
ter Electronic Particle Couter Model ZBI
Hemacytometer counts taken
density, f and g relative
by students.
fluorescence
DESIGN CLASSIFICATION
570
590
632
340
280
180
180
234
240
34
26
27
12
12
0.
2.
1.
2.
2.
HIGH PRODUCTIVITY
(6.10-20.00 mg dry weight
I"1)
MODERATELY HIGH PRODUCTIVITY
(0.81-6.00 mg dry weight
MODERATE PRODUCTIVITY
(0.11-0.80 mg dry weight
5
6 LOW PRODUCTIVITY
5
(0.00-0.10 mg dry weight
2
3
. . Cell suspensions were prepared by EPA
1 >)
I"1)
n>
ALGAL
Calculated dry weights based on student-derived
units of chlorophyll a non-extracted Selenastrum
cells.
-------
a = confidence interval of 66.6 percent
2a = confidence interval of 95.0 percent
3a = confidence interval of 99.0 percent
/Ix2' - (Ix)2/n
CT = ± v - n~n —
Example of calculation—Taking data (dry weight) from Table 4 for the trip-
licate set of control flasks for day fourteen, the constants in the equation
are as follows:
Xl = 0. 14 xx2 = 0.0196
x2 = 0. 14 x22 = 0.0196
x3 = 0.13 xs2 = 0.0169
n = 3
Therefore: Ix2 = 0.0561
(Ix)2 = (0.41)2 = 0.1681
a = ± V(0.0561) - (0.0560) = V0.0005 = ± 0.007
2
2a = ± 0.014
3a = ± 0.021
30
-------
TABLE 4
TYPICAL REPORT OF ASSAY RESULTS
calculated dry wei
control
days
0
3
5
7
10
14
1
.02
.09
.13
.13
.14
.14
2
.02
.07
.13
.12
.13
.14
3
.02
.08
.11
.11
.12
.13
Avg
.02
.08
.12
.12
.13
. 14
control +
1
0.02 0.
1.62 1.
6.10 6.
7.60 7.
8.75 8.
8.80 8.
ght mg 1
0.05 mg
2 3
02 0.02
61 1.56
50 6.61
75 7.65
80 8.70
85 8.75
I-1
P I-1
Avg
0.02
1.60
6.40
7.67
8.75
8.80
control + 1
1
.02
.10
.12
.14
.14
.13
2
.02
.08
. 14
.16
.15
.14
. 0 mg
3
.02
.10
.13
.16
.15
.16
N I-1
Avg
.02
.09
.13
.15
.15
.14
The following is an example of how one determines the required number of
replicates: Considering the design of an experiment to compare two media, one
of known strength (mx) which will produce a maximum standing crop of about
8.75 mg dry weight I-1 and another medium (m2) expected to produce a greater
standing crop. The "null hypothesis," which one expects to disprove, is that
m2 ^ m-L, i.e., that the unknown medium produced a standing crop not larger
than the known medium. The "alternative hypothesis," which one expects to
prove, is that m2 > ml, i.e., that the unknown medium produces a greater
standing crop than the known medium.
How many replicate flasks should be used? The answer can be found by
first answering the following five questions and then consulting Table 5.
Question 1: "What significance level, a, should be used?" For this
example we shall use the significance level a = 0.05, i.e., if the two media
are the same strength (mx = m2) there will be one chance in twenty that the
experiment will result in the erroneous conclusion that the known medium is
weaker (mx < m2).
31
-------
Question 2: "What is the smallest difference, 6 = m2 - mt which must be
detected?" The known medium will produce a standing crop of about mt = 8.75
mg dry wt. I-1. Suppose the other medium must produce a 10% greater crop
(9.62 mg dry wt. I-1) to be "significantly" stronger, i.e., the smallest
difference which must be detected is about 6 = m2 - ml = 9.62 - 8.75 = 0.87 mg
dry wt ~\-1.
Question 3: "With what probability must a difference of 6Q (= 0.87 mg
dry wt I-1) be detected by the experiment?" Suppose a probability of detec-
tion of 0.90 is desired, i.e., if the true difference in the standing crops of
the media is 0.87 mg dry wt I-1. There is a 90 percent chance the experiment
will detect the difference (lead to a conclusion that the known medium is
weaker). Conversely, there is a 10% chance that the experiment will fail to
detect a difference of 0.87 mg dry wt I-1. Denote the probability of detec-
tion as 1 - p = 0.90.
Question 4: "What is the standard deviation, s, of an individual obser-
vation?" (Note that this is not the same as the standard error of a mean of
several observations.) There would probably be some information about the'
standard deviation from a prior experiment with the "known" medium. For this
example assume that previous experience indicates a standard deviation ap-
proximately 0.40 mg dry wt I-1.
Question 5: "Does the alternative hypothesis specify a 'one-tail1
alternative (6 > 0, m2 > mx) or a 'two-tail' alternative (6 ^ 0, m2 ^ mi)?"
In this example it is assumed the findings will be significant only if the
unknown medium produces a greater standing crop than the known medium; thus
the alternative hypothesis specifies a one-tail alternative 6 > 0, m2 > n^.
32
-------
TABLE 5
AID IN COMPUTING SAMPLE SIZES REQUIRED TO DETECT
PRESCRIBED DIFFERENCES BETWEEN AVERAGES
Notation:
a Significance level of the test
6Q Smallest detectable or significant difference
1-p Probability of declaring 6^0 if 6 = 6
s Sample estimate of the standard deviation of an observation
d = 0.7071 6 / s
o
ONE TAIL TEST TABLES:
For a = .01 add 2 to the tabled value to get the number of replicates;
for a = .05 add 1 to the tabled value to get the number of replicates.
a = .01
1-p
d
.1
.2
.4
.6
.8
1.0
1.2
1.4
1.6
1.8
2.0
3.0
.50
542
136
34
16
9
6
4
3
3
2
2
1
.60
666
167
42
19
11
7
4
4
3
3
2
1
.70
813
204
51
23
13
9
6
5
4
3
3
1
.80
1004
251
63
28
16
11
7
6
4
4
3
2
.90
1302
326
82
37
21
14
10
7
6
5
4
2
.95
1578
395
99
44
25
16
11
9
7
5
4
2
.99
2165
542
136
61
34
22
16
12
9
7
6
3
a = .05
1-P
d
.1
.2
.4
.6
.8
1.0
1.2
1.4
1.6
1.8
2.0
3.0
.50
271
68
17
8
5
3
2
2
2
1
1
1
.60
361
91
23
11
6
4
3
2
2
2
1
1
.70
471
118
30
14
8
5
4
3
2
2
2
1
.80
619
155
39
18
10
7
5
4
3
2
2
1
.90
857
215
54
24
14
9
6
5
4
3
3
1
.95
1083
271
68
31
17
11
8
6
5
4
3
2
.99
1578
395
99
44
25
16
11
9
7
5
4
2
33
-------
TABLE 5 (continued)
TWO TAIL TEST TABLES:
For a = .01 add 2 to the tabled value to get the number of replicates; for
a = 0.05 add 1 to the tabled value to get the number of replicates.
a = .01
1-p
d
.1
.2
.4
.6
.8
1.0
1.2
1.4
1.6
1.8
2.0
3.0
.50
664
166
42
19
11
7
5
4
3
3
2
1
.60
801
201
51
23
13
9
6
5
4
3
3
1
.70
962
241
61
27
16
10
7
5
4
3
3
2
.80
1168
292
73
33
19
12
9
6
5
4
3
2
.90
1488
372
93
42
24
15
11
8
6
5
4
2
.95
1782
446
112
50
28
18
13
10
7
6
5
2
.99
2404
601
151
67
38
25
17
13
10
8
7
3
If we must estimate a from our sample and use Student's t, then we
should add 4 to the tabulated values to obtain the approximate required
sample size. (If we are comparing two product averages, add 2 to the tab-
ulated values, to obtain the required size of each sample. For this case, we
must have a. = aR.)
AD nr
a = . 05
1-P
d
. 1
.2
.4
.6
.8
1.0
1.2
1.4
1.6
1.8
2.0
3.0
.50
385
97
25
11
7
4
3
2
2
2
1
1
.60
490
123
31
14
8
5
4
3
2
2
2
1
.70
618
155
39
18
10
7
5
4
3
2
2
1
.80
785
197
50
22
13
8
6
5
4
3
2
1
.90
1051
283
66
30
17
11
8
6
5
4
3
2
.95
1300
325
82
37
21
13
10
7
6
5
4
2
.99
1838
460
115
52
29
19
13
10
8
6
5
3
u + (z-]_a+-i_o)2/d2, where z denotes the cumulative distribution function
of the standard normal (0,1) distribution.
Source: Experimental Statistics, by Mary G. Natrella, National Bureau of
Standards Handbook 91, U. S. Government Printing Office, Washington, DC. The
tables above are Tables A-9 and A-8, respectively, from this reference.
34
-------
Therefore, a one-tail test (Table 5) would be used. (A two-tail alternative
would require a two-tail test.)
In summary, the answers to the questions above have provided the fol-
lowing values:
(1) a = 0.05 = significance level
(2) 6Q = 0.87 mg dry wt. I-1 = smallest "significant" difference
(3) 1 - p = 0.90 = probability of detecting smallest significant dif-
ference.
(4) s = 0.40 mg dry wt I-1 = standard deviation
(5) Alternative hypothesis specifies a one-tail test.
We can now compute the value of "d" and find the required
number of replicates from Table 5:
d = 0.7071 6 /s
o
= (0.7071) x (0.87)7(0.40)
= 1.54
Entering the One-Tail test tables with these values we find the number of
replicates should be between 5 + 1 (corresponding to d = 1.4) and 4 + 1 (cor-
responding to d = 1.6)*- One should use quadratic interpolation in the table,
but linear interpolation produces an approximate result: 6 replicates. Note
that only 4 replicates would have the desired probability of detecting the
difference if d = 2.0, i.e., if 6Q = ~ = - - 1-13. That is, 4
replicates would have a 90% chance of detecting a difference of 1.13 mg dry
wt. I-1, a 13% increase in standing crop, whereas 6 replicates are required to
* Note that the tabled value is not the number of replicates; one must add
1 to the tabled values in the a = 0.05 table and 2 to the tabled values
in the a = 0.01 table.
35
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ensure a 90% chance of detecting a 10% increase in standing crop. These
figures assume the validity of the estimate of the standard deviation.
9.3 Rejection of outliers--An "outlier" among replicate observations is
one whose deviation from the mean is far greater than the rest in absolute
value and perhaps lies three or four standard deviations or further from the
mean. The outlier is a peculiarity and indicates a data point which is not at
all typical. It follows that an outlier should be submitted for particularly
careful examination to see if the reason for its peculiarity can be deter-
mi ned.
Rules have been proposed for rejecting outliers, i.e., for deciding to
remove the observation(s) from the data, after which the data are re-analyzed
with these observations. Automatic rejection of outliers is not always a wise
procedure. Sometimes an outlier is providing information which other data
points cannot since it arises from an unusual combination of circumstances
which may be of vital interest and requires further investigation rather than
rejection. As a general rule, outliers should not be rejected out-of-hand
unless they can be traced to specific causes, e.g., errors in recording ob-
servations or in setting up apparatus. Otherwise, careful investigation is in
order. (The above was adapted from section 3.8 of Applied Regression Analysis
by N. R. Draper and H. Smith, John Wiley and Sons, 1968.)
The following test may be applied for rejecting outliers:
1. Rank order the data in the group containing the outliers (all
observations in the group are supposedly treated alike):
Xi ^ X2 ^ . . .X
12 n
2. Compute the appropriate criterion:
36
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X2 ~
If K! is the outlier c =
x - xx
x -(x -
If xn is the outlier c = n Xn
n x - Xi
n -1
3. If c exceeds the critical value opposite "n" in Table 6, reject the
out! ier.
TABLE 6
CRITICAL VALUES FOR DETERMINING OUTLIERS
n
a
3
4
5
6
7
Example—Suppose
= 0.05
0.941
0.765
0.642
0.560
0.507
the fol
Critical values
a = 0.01
0.988
0.889
0.780
0.698
0.637
lowing replicate dry wt mg I-1 obs
tions were made: 9.8, 4.7, 8.4, 8.0, 8.4, and 7.9. The value 4.7
is suspected to be an outlier. Rank order the data.
Xl x2 x3 x4 x5 x6 n = 6
4.7 7.9 8.0 8.4 8.4 9.8
The criterion is as follows:
X2 " Xl 7.9 - 4.7 _ 3.2
c =
x - x, 9.8 - 4.7 5.1
n
= 0.63
Since n = 6, this value is significant at the a = 0.05 level (0.63 >
0.560), but not at the a = 0.01 level (0.63 < 0.698).
The experimenter who is willing to discard 5% of all his good
data would discard the observation 4.7 as an outlier. The experi-
menter who is willing to discard only 1% of his good data would keep
37
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the observation unless he can determine an experimental reason for
rejecting it.
4. If there are two suspected outliers (say x{ and xn or xx
and x2), the test may be repeated; apply it to the "worst" outlier
first.
5. Note that the regular use of this procedure will result in
discarding five percent (if a = 0.05) or one % (if a = 0.01) of all
one's good (valid) observations.
10.0 EXPERIMENTAL DESIGN AND ANALYSIS
10.10 Nutrient limitation
10.11 Introduction—The "Algal Assay: Bottle Test" can be used to
define nutrient limitation in natural waters, whether this limitation is due
to nitrogen, phosphorus or trace element deficiency. This is accomplished by
an experimental design which incorporates an internal check and balance
system centered around the growth response of ^5. capricornutum to singular and
combined additions of nitrogen, phosphorus, and EDTA to the test waters. The
growth responses obtained are then evaluated to ascertain the limiting nu-
trient(s).
10.12 Experimental design—The following series of nutrient and
chelator additions in Table 7 are considered as the minimum necessary to
determine the nutrient status of an unevaluated test water. They are: the
test water control and final spike concentrations equivalent to mg I-1 in
each test flask.
38
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TABLE 7
BASIC EXPERIMENTAL DESIGN TO DEFINE NUTRIENT LIMITATION
Control
Control + 0.05 mg P I-1 as K2HP04
Control + 1.00 mg N I-1 as NaN03
Control + 0.05 mg P I-1 + 1.00 mg N I-1
Control + 1.00 mg Na2 EDTA I-1 as Disodium (Ethylenedinitrilo)
tetraacetate
Control + 0.05 mg P I-1 + 1.00 mg Na2 EDTA I-1
Control + 1.00 mg N I-1 + 1.00 mg Na2 EDTA I-1
Control + 0.05 mg P I-1 + 1.00 mg N I-1 + 1.00 mg Na2 EDTA I-1
Each nutrient chelator addition was selected based on past experience of
evaluation effectiveness. For example: the 0.05 mg P I-1 spike was chosen to
insure the saturation (excess) of phosphorus within the sample, which is
necessary to drive the system to the secondary limiting nutrient. Each |jg P
I-1 will support 0.430 ± 20% mg dry weight I-1 of S. capricornutum if other
constituents are not growth limiting. Therefore, the 0.05 mg P I-1 additions
should support additional growth in the control test water up to a maximum of
21.50 mg dry wt I-1 depending upon the availability of other essential nu-
trients (primarily nitrogen) within the test water.
Similar rationale pertains to the selected nitrogen addition of 1.0 mg
I-1 which should support an additional increase in biomass up to 38.00 mg dry
wt I-1 (0.038 ± 20% mg dry wt per mg N 1-1) or to that level which can be sup-
ported by (in most cases) the available phosphorus content in the test water.
The combined nitrogen and phosphorus addition will generally support
growth relative to the phosphorus content in the water. This reflects the
39
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excess nitrogen conditions -which are intentionally introduced into the test
water, i.e., 38.00 mg dry wt I-1 for the nitrogen spike versus 21.50 mg dry wt
I-1 due to the phosphorus addition.
The Na2 EDTA chelator addition of 1.00 mg I-1 was selected after the
evaluation of additions of 0.3, 1.0, 5.0 and 10.0 mg I-1 upon the growth
response of S. capricornutum in both assay medium and selected natural waters.
The lowest addition (0.3 mg I-1) was capable of insuring trace element avail-
ability in the culture medium (see sec. 5.0) but was not sufficient to complex
the heavy metals present in many natural waters. Na2 EDTA addition in excess
of 1.0 mg I-1 caused complexion of essential macronutrients (i.e. Ca and Mg)
depressing growth relative to the N and P content of the test waters.
10.13 Essential background data--The mim'mum chemical data neces-
sary to evaluate the assay response to define nutrient limitation are: Ini-
tial pH; Total phosphorus; Ortho-P; N02; N03; NH3 and total Kjeldahl nitrogen.
10.14 Test conditions — Each test flask is inoculated to contain a
final concentration of 1,000 cells ml-1 of S. capricornutum and is incubated
at 24 ± 2°C under 4304 lumens (400 ± 10% ft-c) and shaken once daily or
continuously (see sec. 4.21) for a period of at least 14 days.
10.15 Interpretation of results--Al_[ nutrient limitation assay
results must be reported as the maximum standing crop (MSC) in mg dry wt I-1.
Typical 14-day growth responses representative of phosphorus, nitrogen, trace
element and nitrogen plus phosphorus growth limitation are presented.
10.16 Phosphorus 1 imitation—The following growth responses (Table
8A) and the corresponding control test water chemical analysis data (Table 8B)
are typical of phosphorus-limited waters.
40
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TABLE 8A
GROWTH RESPONSES REPRESENTATIVE OF PHOSPHORUS LIMITATION
Sample Treatment MSC (mg dry wt I-1)
Control 2.16
Control + 0.05 mg P I-1 5.81
Control + 1.00 mg N I-1 2.30
Control + 1.00 mg N and 0.05 mg P I-1 23.69
Control + 1.00 mg Na2 EDTA I-1 2.10
Control + 1.00 mg Na2 EDTA + 0.05 mg P I-1 5.66
Control + 1.00 mg Na2 EDTA + 1.00. mg N I-1 2.30
Control + 1.00 mg Na2 EDTA + 0.05 P + 1.00 mg N I-1 24.60
TABLE 8B
CHEMICAL ANALYSIS OF THE PHOSPHORUS LIMITED CONTROL TEST
WATER AND PREDICTED N AND P YIELDS (mg I-1).
0.021 mg Total P I-1
0.006 mg Ortho-P I-1 = 0.006 x 430 = 2.58 ± 20%*
0.368 mg Total N I-1
0.120 mg N03 + N02-N I-1
0.040 mg NH3-N I-1
0.160 mg TSIN-1 (N02 + N03 + NH3) = 0.160 x 38 = 6.10 ± 20%*
>26:1 N:P ratio (TSIN -=- Ortho-P)
* Predicted yields of S. capricornutum based on soluble inorganic nitrogen
or phosphorus content of the test water if all other essential nutrients are
present in excess.
The ratio of the TSIN to Ortho-P yield factors (38 and 430, respectively)
indicates an optimum N:P ratio of s 11:1 for the support of S. capricornutum.
The N:P ratio can be used as a "guide" to nutrient limitation in most natural
41
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waters. That is, waters containing N:P ratios greater than 11:1 may be con-
sidered phosphorus limited while those containing N:P ratios less than 11:1
can be considered nitrogen limited for algal growth. Placement into a ni-
trogen or phosphorus limitation category without actual assay analysis ij;
discouraged. Only assay response to the nutrient and/or chelator additions
can verify nutrient limitation and the extent of N and P bioavailability in
the test water. The test water used in this example has a N:P ratio of >
26:1. This strongly indicates the potential for phosphorus limitation. The
actual assay response confirms the N:P ratio prediction of nutrient limitation
in this test water.
Differences in maximum standing crop are not considered statistically
different at the 95% (2a) confidence level if they fall within the limits
established in Table 1. Therefore, only the responses obtained by addition of
phosphorus, singly and in combination, with nitrogen and Na2 EDTA are con-
sidered to be statistical1y significant in this test water. These responses
are directly proportional to the increase in phosphorus, and are secondarily
limited by the TSIN content of the test water. For example: 0.160 mg N I-1
contained in the control test water can support 6.10 ± 20% mg dry wt I-1 of S.
capricornutum due to its nitrogen availability, even though the addition of
0.05 mg P I-1 was enough phosphorus to support 21.50 mg dry wt I-1.
The phosphorus regulated growth response obtained in the control and in
the test waters containing additions of nitrogen and Na2 EDTA, singly and in
combination, should be essentially identical (within ± 20%) in the phosphorus
limited test waters. Thus, 12 replicate flasks can be used as built-in check
and balance criteria to define the validity and accuracy of the assay results.
For example: if the yield in any of these replicate flasks exceeded ± 50% it
42
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would immediately be suspect as an outlier and in most cases would be dis-
carded.
The biological availability of nitrogen and phosphorus in the test water
can be calculated by dividing the MSC by either the TSIN or Ortho-P yield
factors. The MSC obtained with 0.05 mg P I-1 addition should be used to
calculate nitrogen availability. For example: 5.81 -=- 38 = 0.152 mg available
nitrogen I-1. This calculated value compares favorably with the chemically
analyzed TSIN value of 0.160 mg I-1. Thus, in this test water all of the TSIN
was available for growth of the test alga. This conclusion is important
because no other growth factor except phosphorus in the presence of adequate
nitrogen is regulating growth in this test water.
The biologically available phosphorus content in this test water is
derived by dividing the yield obtained with 1.00 mg N I-1 addition by the
phosphorus yield coefficient. Thus, the control plus 1.00 mg H I-1 yield of
2.30 -=- 430 = 0.005 mg available P I-1. This back calculated value of 0.005 mg
I-1 is verification of the chemically analyzed value of 0.006 mg Ortho-P I-1.
This biologically reactive phosphorus value (0.005 mg I-1) can also be used to
calculate the percentage of bioavailable total phosphorus (0.021 mg I-1)
which in this test water is 24% (0.005 -f 0.021). The bioavailable nitrogen
and phosphorus concentrations in this test water correlate with their chem-
ically analyzed concentrations. Failure of a test water to attain this corre-
lation can be attributed to: presence of bioavailable organic nutrients;
effect of other growth-1imiting nutrients; the presence of inhibitory con-
stituents in the test water; and/or unreliable chemical analysis for Ortho-P
and TSIN.
43
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10.17 Nitrogen 1imitatiorr-The following assay growth responses
(Table 9A) and corresponding control test water chemical analysis data (Table
9B) are typical of nitrogen limitation in natural test waters.
TABLE 9A
GROWTH RESPONSES REPRESENTATIVE OF NITROGEN LIMITATION
Sample Treatment MSC (mg dry wt I-1)
Control 4.06
Control + 0.05 mg P I-1 4.21
Control + 1.00 mg N I-1 12.68
Control + 1.00 mg N + 0.05 mg P I-1 34.52
Control + 1.00 mg Na2 EDTA I-1 6.30
Control + 1.00 mg Na2 EDTA + 0.05 mg P I-1 6.49
Control + 1.00 mg Na2 EDTA + 1.00 mg N I-1 12.80
Control + 1.00 mg Na2 EDTA + 1. 00 mg N + 0. 05 mg P I-1 34.68
TABLE 9B
CHEMICAL ANALYSIS OF THE CONTROL TEST WATER AND CORRESPONDING
N:P RATIO WITH PREDICTED YIELDS (mg I-1).
0.072 mg Total P I-1
0.030 mg Ortho-P I-1 = 0.030 x 430 = 12.90 ± 20%
0.160 mg Total N I-1
0.055 mg N03 + N02-N I-1
0.020 mg NH3-N I-1
0.075 mg TSIN I-1 = 0.075 x 38 = 2.85 ± 20%
2.5:1 N:P ratio (TSIN v Ortho-P)
The growth responses obtained in the control and the control plus ni-
trogen and/or chelator additions identify nitrogen as the primary growth
44
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limiting nutrient. These responses can also be used to define: the bioavail-
able concentrations of nitrogen and phosphorus; chemical analysis reliability;
and nitrogen form utilization.
The following basic assay response analyses were used to define the
critical nutrient interactions regulating growth in the test water. The 12.68
mg dry wt I-1 obtained by the addition of 1.0 mg N I-1 confirms the N:P ratio
(2.5:1) limiting nutrient status assigned to this test water. This nitrogen
stimulated maximum standing crop divided by the phosphorus yield factor (12.68
-r 430 = 0.029 mg I-1) indicates the bioavailable phosphorus content of the
test water. The resultant bioavailable concentration of 0.029 mg P I-1 is
essentially identical to the Ortho-P content of the test water (0.030 mg P
I-1).
The bioavailable nitrogen content of the test water was determined by
dividing the phosphorus stimulated response by the nitrogen yield factor (4.21
~ 38 = 0.111 mg N I-1). This bioavailable nitrogen concentration is 1.5 fold
greater than the analyzed TSIN content of the test water. The increase in
available nitrogen may be attributed to: unreliable chemical analysis; the
utilization of other nitrogen forms (such as organic nitrogen) for the support
of S. capricornutum; or unreliable assay test results.
The built-in check and balance response yield relationships to the
recommended nutrient and/or chelator additions, can be used to define the
validity of the calculated 1.5 fold increase of bioavailable nitrogen content
in the test water. The first check and balance evaluation is to determine
whether the assay yields obtained in the control test water are "statistically
equal" to those obtained in the control plus phosphorus test water. The
.rationale being that; growth in nitrogen limited waters should not be respon-
45
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sive to singular addition of phosphorus. Thus similar yields of 4.06 and 4.21
mg dry wt I-1 attained in these test waters confirms the reproducibility and
precision of the assay test results. Therefore, the "statistically signif-
icant" response of 12.68 mg dry wt I-1 obtained by nitrogen addition to the
test water (proportional to its bioavailable phosphorus content) validates the
primary nutrient limitation status of the test water.
The second algal assay response evaluation to be considered is the
identification of the secondary growth-regulating nutrient(s) in the test
water. This is accomplished by defining the comparability between the yields
obtained in the combined nitrogen and phosphorus spike with those attained in
the combined nitrogen, phosphorus, Na2 EDTA spiked test water. The response
of the test alga to combined N and P addition should be "statistically equal"
(within ± 10%) to the yield obtained with N, P and Na2 EDTA addition if a
trace-element is not growth limiting. The similar yields obtained of 34.52
and 34.68 mg dry wt I-1 respectively, strongly indicate that the growth re-
sponse is regulated solely by the N and P content in the test water. The
comparison of these assay yields with those calculated from the TSIN and
Ortho-P content of the test water should identify the secondary growth-regu-
lating nutrient. The TSIN and Ortho-P calculated yields for these test waters
are:
TSIN yield = the TSIN content of the test water (0.075 mg I-1) plus
that added in the spike (1.00 mg N I-1) multiplied by the nitrogen yield
factor (38 x 1.075 = 40.85 ± 20% mg dry wt I-1) equals the MSC which can
be supported in the test water.
Ortho-P yield = 0.030 mg P I-1 in the control plus 0.050 mg P I-1 in
the spike multiplied by 430 (0.080 x 430 = 34.40 ± 20% mg dry wt I-1)
46
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indicates that a MSC of 34.40 ± 20% mg dry wt I-1 can be supported by the
phosphorus content of the test water. The MSC obtained by assay analysis
of 34.52 and 34.68 mg dry wt I-1 to combined N and P additions are sta-
tistically equal to those calculated for the phosphorus content in the
test waters. Therefore, phosphorus is the secondary growth regulating
nutrient.
By evaluating these assay responses we have established: (1) nitrogen is
primarily regulating growth; (2) the precision and reproducibility of the
assay; (3) phosphorus addition in the presence of excess nitrogen supports
growth to its maximum potential; (4) the absence of other growth regulating
constituents; and (5) an apparent increase in nitrogen availability beyond
that attributed to the TSIN content of the test water.
Establishing that the growth response in a test water j_s not regulated by
an unknown trace-element or inhibitor is prerequisite to defining the relia-
bility of the chemical analysis of TSIN in nitrogen limited waters. This is
partly due to the ability of the test alga to metabolize the Na2 EDTA complex
in the presence of associated bacteria. This is important not only in studies
of trace-element limitation (discussed in subsection 10.18) but also suggests
the possible utilization of the nitrogen contained in the complex to support
growth as well.
The standard addition of 1.00 mg Na2 EDTA I-1 contains 0.075 mg N I-1.
If this nitrogen is bioavailable it would support an additional 2.85 ± 20% mg
dry wt I-1 increase in S. capricornutum standing crop (0.075 x 38 = 2.85).
The additional response obtained with Na2 EDTA addition over that in the
control was 2.24 mg dry wt I-1. Similar additional response (2.43 mg dry wt
47
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I-1) was obtained in the combination Na2 EDTA plus phosphorus spiked test
water (6.40 - 4.06).
These growth responses suggest that Na2 EDTA may have been metabolized
and that growth was obtained relative to its nitrogen content. This response
also indicates the possible utilization of organic bound nitrogen fractions in
the test waters as growth stimulators. Thus, the 1.5 fold increase in cal-
culated bioavaiTable nitrogen may be due to organic nitrogen utilization
rather than to unreliable TSIN chemical analysis.
The algal responses to this representative nitrogen limited test water
were chosen to identify all of the possible nitrogen interactions that can
regulate growth of Jj. capricornutum assayed in accordance with the prescribed
test protocol. The metabolism of Na2 EDTA and the subsequent utilization of
its nitrogen content for support of additional growth has been defined j_n
less than J% of all nitrogen limited natural waters studied by this labora-
tory.
10.18 Trace-element 1imitation--Trace-element limitation is rare in
most natural waters. Less than 2% of the 150 natural waters investigated by
this laboratory were trace-element growth regulated. Growth in these trace-
element limited waters was most often limited by the availability of iron.
Synthetic organic ligands such as Disodium (ethylenedinitrilo) tetra-
acetate (Na2 EDTA) are added to defined inorganic culture media to make sure
trace elements, principally Fe and Mn, are available to support algal growth.
Recognition of the growth enhancement qualities of organic ligands led to
addition of Na2 EDTA to natural test waters prior to the assay to ascertain
trace-element availability.
48
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Columbia River water, collected at Rock Island Dam and Bridgeport, Wash-
ington, was identified as being trace-element deficient (Miller, Greene,
Shiroyama, 1976a). The N:P ratios of these waters of 9:1 indicated potential
nitrogen growth limitation, as did the theoretical yield predictions based on
Ortho-P and TSIN content. The theoretical yield (±20%) for Columbia River
water collected at Rock Island Dam based on TSIN content of 0.109 mg I-1 is
4.10 mg dry wt I-1 of the test alga. This water supported less than 10 per-
cent of the predicted yield in the control, or in the control plus nitrogen or
phosphorus added singly or in combination. The addition of 1.00 mg Na2 EDTA
I-1, however, stimulated growth to 5.40 mg dry wt I-1, 128 percent of the
predicted control yield. The addition of Na2 EDTA may have increased iron
availability, thus stimulating growth. The concentrations of total soluble
ferric iron that can be in equilibrium with ferric hydroxide at pH 8.0 in
oxygenated water is approximately 0.2 ug I-1. The iron requirement for
optimum growth of ^. capricornutum is 4.5 pg I-1, 22.5 times greater than the
normal concentration in soluble form. Addition of Na2 EDTA stabilizes soluble
iron availability in natural waters. Theis and Singer (1973) stated that the
exact mechanism(s) by which organic ligands interact with iron are not known.
Their research has shown that organic ligands, such as EDTA, can stabilize
ferrous iron through the formation of organic complexes which are resistant to
oxygenation in natural waters, thus increasing the availability of iron for
aquatic growth. Barber (1973) studied growth enhancement effects of EDTA
addition to sea water. He concluded that organic ligands may increase the
mobility of essential metals such as Fe and Mn, but that these findings do
not limit the possibility that organic ligands enhance phytoplankton growth by
suppressing heavy metal toxicity. Without comprehensive trace-metal analysis
49
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no judgment may be made as to whether algal growth is limited by either sub-
optimal or toxic trace-metal content of the water (Miller, Greene, and Shiro-
yama, 1976b).
10.19 Nitrogen and phosphorus co-1 imitation—Nitrogen and phos-
phorus co-limitation is most commonly observed in high nutrient (eutrophic)
waters. An analysis of the N:P ratios in these highly productive waters is
usually sufficient to assess co-limitation conditions. N:P ratios ranging
between 10 and 12:1 generally indicate possible co-limitation. Actual assay
verification, using the nutrient and/or chelator additions outlined in section
10.12, is necessary to establish the nutrient limitation status of a test
water. Growth response to the singular addition of nitrogen, phosphorus and
Na2 EDTA will be essentially identical (within ±20%) in N and P co-limited
waters. Significant increase in growth response wil1 only b^ obtained in the
combined N and £, as well as in the N and P, and Na2 EDTA combination, spiked
test waters.
10.20 Practical application of nutrient limitation studies—Understand-
ing of the interaction of nutrient dynamics and its regulation of aquatic
productivity in natural waters is necessary to establish sound management
alternatives.
No singular chemical test or biological measurement (i.e., Ortho-P or
chlorophyll a) can be used to define all the interactions regulating bio-
logical productivity in natural waters. However, the "Algal Assay: Bottle
Test" (AA:BT) can be used to define and/or predict the nutrient availability
in most natural waters. This test can also identify and/or predict the algal
growth potential of natural waters. For example: The AA:BT must be used to
assess the trophic status of a natural water. In most cases the placement of
50
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a test water into a trophic category is based on the bioavaiTable nitrogen and
phosphorus content of a test water. Those waters containing greater than
0.015 mg bioavailable P I-1 and 0.165 mg bioavailable N I-1 are considered
eutrophic. In general, these values correlate to the Ortho-P and TSIN chem-
ical analysis content in the test waters. Test waters in which the assay
response does not correlate with their available N and P content may be trace-
element growth regulated. As this is quite rare, toxicity is usually indi-
cated in these test waters.
The AA:BT nutrient limitation experimental design growth responses can be
used to define the effectiveness of the following management alternative:
What is the effectiveness of an 80% reduction of domestic waste
phosphorus loading upon receiving water quality?
The AA:BT can verify the bioavailable phosphorus content of the
wastewater; the post treatment available P content in the receiving
water; and the interaction of other nutrients affecting biological
productivity in the receiving water. For example: A domestic waste
containing 7.5 mg Total PI-1 and 27.0 mg Total N I-1 is discharged into
a receiving water. This treatment plant discharge contributes 60% of the
total phosphorus and 40% of the total nitrogen to the receiving water.
The downstream receiving water has an average total phosphorus content of
0.060 mg I-1, 60% of which (0.036 mg I-1) is due to the waste inflow.
The remaining 40% phosphorus content (0.024 mg I-1) is contributed up-
stream from the treatment plant.
The assay response in the receiving water downstream from the facil-
ity outfall, before treatment of the domestic waste to reduce phosphorus
loading, was 11.18 mg dry wt I-1. The bioavailable phosphorus concen-
51
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tration needed to support this yield is 0.026 mg P I-1, (11.18 4- 430 =
0.026) which is 43% of the receiving water (0.060 mg I-1) total phos-
phorus content.
The 80% reduction in total phosphorus loading should result in a
final total phosphorus receiving water concentrations of 0.031 mg P I-1.
[0.060 - 0.036 + (20% x 0.036) = 0.031]
where: 0.060 = mg I-1 downstream P concentration
0.036 = mg P I-1 contributed by treatment plant
20% x 0.036 = concentration of post treatment P I-1 contri-
bution by treatment plant operating at 80%
efficiency.
Assuming that a similar percentage (43%) of the post treatment
receiving water total P is bioavailable, one would predict that a biomass
of 5.78 mg dry wt I-1 ( a reduction of 52%) would be obtained in the
receiving water [430 (43%) x 0.031) = 5.78].
The AA:BT results suggest that:
(1) The 52% anticipated reduction in algal growth is still considered a
eutrophic condition.
(2) A water use cost benefit analysis should be conducted before treat-
ment is initiated.
(3) Phosphorus removal may only be necessary during peak growth condi-
tions (July, August, September).
(4) Reduction of upstream phosphorus loading in conjunction with ad-
vanced wastewater treatment would vastly improve water quality.
Similar experimental design and analysis rationale can be used to define
and help solve other water quality management problems such as:
52
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(1) Determine the feasibility of nutrient criteria, i.e., establishing
a 1.0 mg total P I-1 effluent standard.
(2) Define the "real world" impact of land use upon nutrient loading to
receiving waters.
(3) Define and monitor the effectiveness of established effluent guide-
lines.
(4) Determine the effluent criteria for specific complex wastes based on
their stimulatory or inhibitory properties.
10.30 Heavy metal toxicity
The ability of the "Algal Assay:Bottle Test" to predict the algal growth
potential of lakes and streams and its use to define limiting nutrients in
these natural waters led to the identification and application of nitrogen and
phosphorus yield factors to predict the growth of S. capricornutum (section
10.10; Nutrient limitation).
Failure of a test water to attain the predicted yield or nutrient limi-
tation (N, P, trace-element) status when assayed in accordance with the
experimental design protocol outlined in subsection 10.12 usually indicates
the presence of toxicants. The AA:BT can be used to define the interactions
of heavy metals upon productivity within aquatic ecosystems.
The study of heavy metal interaction in natural waters is complicated by
an uncertainty of the form, concentration, and biological reactive state of
the metal. Thus, with few exceptions, the chemically analyzed heavy metal
content of a test water may not reflect the resultant biological interactions
and productivity in natural waters. The growth response of S. capricornutum
to conditions of heavy metal stress in natural waters is in essence a "bio-
logical response model" of complex physical and chemical interactions. The
53
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resultant biological response (maximum standing crop) is an integration of the
combined effects of solubility, ionic strength, metal concentration, and
contact time which regulate toxicity of the heavy metal to the test organism.
10.31 Experimental design—The basic experimental design to deter-
mine the extent of heavy metal toxicity and its interaction upon nutrient
regulation of the test alga in natural waters is shown in Table 10:
TABLE 10
BASIC EXPERIMENTAL DESIGN TO DEFINE HEAVY METAL TOXICITY
Control
Control + 1.00 mg Na2 EDTA I-1
Control + 1.00 mg Na2 EDTA + 0.05 mg P I-1
Control + 1.00 mg Na2 EDTA + 1.00 mg N I-1
Control + 1.00 mg Na2 EDTA + 0.05 mg P and 1.00 mg N I-1
10.32 Essential background data—The minimum chemical data neces-
sary to substantiate the presence of heavy metal toxicity are: Initial pH;
Total phosphorus; Ortho-P; Total Kjeldahl N; N02; N03 and NH3-N. The growth
response of the test algal is compared to the predicted yields based on the
analyzed nutrient content of the test waste or receiving water. Those wastes
or receiving waters which do not support growth within ± 20% of their limiting
nutrient potential are then analyzed for the suspected heavy metals.
10.33 Test condition—Each test flask is inoculated to- contain a
final concentration of 1000 cells ml-1 of S. capricornutum; incubated at 24 ±
2°C under 4304 lumens (400 ± 10% ft-c) and shaken once daily (see section
4.21) or continuously for a period of at least 14 days.
54
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10.34 Interpretation of results—All toxicity results must be
reported as the % inhibition at day 14 (% I14) based on the difference in mg
dry wt I-1 obtained in the control with that produced in the Control test
water containing 1.00 mg Na2 EDTA I-1.
The growth responses shown in Table 11 A, and corresponding control test
water nutrient chemical analysis data (Table 11B), are typical of those ob-
tained in heavy metal contaminated receiving streams.
TABLE 11A
GROWTH RESPONSES REPRESENTATIVE OF HEAVY METAL TOXICITY
Sample Treatment MSC (mg dry wt I-1)
Control 0.12 > 95% I14
Control + 1.00 mg Na2 EDTA I-1 21.70
Control + 1.00 mg Na2 EDTA + 0.05 mg P I-1 20.90
Control + 1.00 mg Na2 EDTA + 1.00 mg N I-1 49.60
Control + 1.00 mg Na2 EDTA + 1.00 mg N + 0.05 mg P I-1 50.20
TABLE 11B
NUTRIENT ANALYSIS OF THE METAL CONTAMINATED CONTROL TEST
WATER AND CORRESPONDING PREDICTED YIELDS (mg I-1)
0.175 mg Total P I-1
0.115 mg Ortho-P I-1 = 0.155 x 430 = 49.45 ± 20%
0.895 mg Total N I-1
0.365 mg N03 + N02-N I-1
0.144 mg NH3-N I-1
0.509 mg TSIN I-1 = 0.509 x 38 = 19.30 ± 20%
4.4:1 N:P ratio (TSIN -=- Ortho-P)
55
-------
The > 95% I14 growth response obtained in this test water is indicative of
heavy metal toxicity. The addition of 1.00 mg Na2 EDTA I-1 to this test water
complexed the bioreactive metals, enabling the test alga to achieve the maxi-
mum nitrogen limited standing crop of 21.70 mg dry wt I-1. The 2.2 fold
increase in maximum yield, beyond that achieved in the chelated control,
obtained with combined chelator and nitrogen addition suggests that nitrogen
is the secondary growth-regulating constituent. The addition of phosphorus to
this nitrogen, chelator combination did not stimulate growth greater than that
predicted for the TSIN content (1.509 x 38 = 57.34 ± 20%) of the test water.
The inhibited growth response obtained in the control test water is
attributed to its analyzed heavy metal content. This test water contained
0.125 mg Zn I-1, 0.006 mg Cu I-1, 0.001 mg Cd I-1, 0.038 mg Al I-1 and 0.009
mg Pb I-1.
These growth responses have established the sensitivity of S. capricorn-
utum to the bioreactive state of these heavy metals. The >95% I14 algistatic
response of the test alga in the control test water is similar to that of
sensitive indigenous species to accidental or recent discharges of heavy
metals (an algicidal response is verified when a subculture from an algistatic
test water fails to grow in assay medium). However, this inhibited response
does not necessarily reflect the growth potential of indigenous algae which
have evolved from long term chronic exposure to heavy metals.
The response of the standard laboratory algal test organism to the addi-
tion of Na2 EDTA, singly and in combination with nitrogen and phosphorus, to
heavy metal laden test waters has been shown to correlate (r = 0.82) with
indigenous phytoplankton standing crop (Greene et a_[., 1978). The indigenous
phytoplankton growth in these waters can be attributed to: (1) adaptation to
56
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their environment; (2) natural decomposition and/or complexing of the heavy
metals by both organic and inorganic ligands; and (3) the presence of adequate
nutrients.
10.40 New product evaluation
10.41 Introduction—The advent of recent Toxic Substance Control
legislation outlined in Public Law 94-469; (October 11, 1976) and the urgent
need to establish test procedures and effluent guidelines for pollutants has
led to a flurry of bioassessment activity. This activity is relevant because
only the bioreactive components of the pollutants are responsible for the
regulation of biological productivity in natural waters.
The continued acceptance of chemical analysis of specific constituents
within the product formulations (i.e., Zn, Cu, Cd, phenol, PCB, aniline) as
the primary reference standard for the legislation of ecological response
criteria is both unwise and misleading. Only concurrent evaluation of both
chemical analysis and bioassay results will provide the scientific base
necessary to establish realistic water quality criteria.
The AA:BT can be used to define the potential stimulatory and/or inhib-
itory properties of new product formulations introduced into receiving waters.
10.42 Experimental design—It is important to consider the fol-
lowing factors when designing an assay experiment to evaluate the environ-
mental impact of new product formulations:
(1) The geographical distribution and intended use of the product.
(2) The method of entry into the receiving water (i.e., direct
discharge or discharge after primary, secondary or advanced
wastewater treatment, etc.).
57
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(3) The recommended application formulation.
(4) The anticipated final concentrations (usage and dilution ra-
tios) of the product within the receiving water.
(5) The degree to which the test waters are representative of those
within the geographical area of product use.
The relative importance of these factors will vary with each specific
product that is evaluated. However, basic experimental design criteria and
rationale are applicable in evaluating all new product formulations.
The following experimental protocol is an example of how the AA:BT is
used to define the potential stimulatory and/or inhibitory impact of new
detergent formulations. The impact of detergent formulations upon aquatic
productivity is most often ascribed to the product's nutrient (primarily
phosphorus) content. Detergent derived-nutrients usually enter receiving
waters as components of domestic waste water effluents. Procter and Gamble
(1976) estimate that 35% of the phosphates in domestic sewage originates from
detergents.
The amount of a candidate detergent formulated product to be added to a
test water can be calculated directly from historical treatment plant phos-
phorus loading curves (i.e., 35% of the phosphorus in sewage x % of waste
loading to receiving water) or from theoretical detergent loading equations
(Hall, 1973).
A sample detergent loading calculation based on 12,000 wash loads per
day, treated and discharged from a treatment plant (15 x 106 liters per day)
into a receiving water containing 854 x 106 liters per day (349 cfs) is as
follows:
58
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Assume:
(1) one cup of detergent (73 x 103 mg) added to each washload.
(2) 30% of the detergent remains after waste treatment.
Therefore: A x |j x C x E = F
where; A = mg detergent per wash load.
B = no. of washloads per day (population -r 3.26 = washloads per day).*
C = % detergent remaining after treatment.
D = liters per day of waste discharge (mgd x 3.79)
E = % treated waste contained in receiving water (liters per day waste
discharge 4- liters per day in receiving water). Note! cfs x 2.448
= 1 x 106 liters per day.
F = mg I-1 of detergent in receiving water.
73 x 103 x 12,000 x 0.30 ni_ . on . ,
15 x 1Qft ^.ters x .017 = 0.30 mg l-i
Thus, in this example, 0.30 mg I-1 of detergent would be contained in the
receiving water mixing zone downstream from the domestic waste discharge.
Receiving waters must be used in the evaluation of new product formu-
lations. These test waters should be collected upstream from the waste
inflow in accordance with the methods outlined in section 3.1.
A typical receiving water assessment should include the calculated
product concentration as well as 0.5 and 5.0 fold (mg I-1) additions, or other
additions as deemed environmentally significant.
A minimum assay evaluation of the test detergent in our example would
include:
(1) Control receiving water(s)
After Hall, 1973.
59
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(2) Control + 0.15 mg detergent I-1
(3) Control + 0.30 mg detergent I-1
(4) Control + 1.50 mg detergent I-1
Assay results obtained from this experimental design will determine the effect
of the material above, but not the effect of the material in addition to or
its interaction with the current wastewater discharge to the receiving stream.
These effects can be obtained by expanding the experimental design to include;
(1) upstream receiving water plus the % wastewater equal to the normal back-
ground level, and (2) wastewater plus the predetermined detergent levels.
The degree of growth stimulation or inhibition of the test material added
to a receiving water is usually defined by dividing the 14-day mg dry wt I-1
MSC in the treated test water by the MSC supported in the control test water.
In some test waters the MSC may not be achieved until after day 14 (see cri-
teria outlined in section 8.3). Maximum standing crop assessment in these
waters should be made at 2-day intervals following day 14 until the maximum
yield is obtained.
Treated: Control ratios < 1.0 indicate inhibition, while ratios > 1.0
suggest stimulation. Stimulation ratios are expressed as statements of the
receiving water product concentration responsible for the stimulation at the
time of maximum yield (e.g., 0.31 mg detergent I-1 = 2.8).
Inhibition responses can be reported as either the % inhibition at the
time in days the MSC is obtained (e.g., %I14) based upon the difference in mg
dry wt I-1 obtained in the control water with that produced in the treated
water, or as the aforementioned Treated:Control ratio, i.e., 0.5. Inhibition
can be either algistatic or algicidal. The subculture of the test alga from
60
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an apparent algistatic test water, into the algal assay medium (section 5.0),
must be used to verify an algicidal response.
Reduction of 50% in MSC (EC50) is not an acceptable toxic response for
assessment of algal growth. Miller, Greene, and Shiroyama (1976), have
reported that the inhibition of specific heavy metals upon the growth of S.
capricornutum may be linear (0 - 100%) with the increase in zinc content of
test waters, but non-linear for the increase in copper and cadmium content
beyond 20 and 40% respectively. Additional increase in either Cu of Cd
resulted in > 95% I14 of the test alga. Payne and Hall (1978), also dis-
courage the use of EC50 response values to define the toxic effects of new
detergent formulations.
10.5 Evaluation of Complex Wastes
10.51 Introduction—Point and/or non-point waste effluents gener-
ated from industrial, agricultural, and domestic treatment and sludge disposal
activities usually contain both inorganic and organic components. The inter-
action of these complex wastes and the extent to which they regulate biologi-
cal productivity in natural waters is not well defined. This is in part due
to past research in which the response of selected test organisms to specific
constituents (i.e., Zn, Cd, Cr, Cu, DDT, PCB, etc.) cultured in defined media
was used to establish toxicity criteria. This concept is faulty because it
does not reflect the antagonistic and/or synergistic interactions of the
organic and inorganic ligands contained within both the complex wastes and the
receiving waters. This shortcoming, coupled with the use of chemical analysis
data as the basis for biological water quality criteria, has caused concern
among regulating agencies.
61
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The AA:BT is centered around the concept that only the bioreactive
components of pollutants are responsible for the regulation of biological
productivity in natural waters. Therefore, the bioassay should be used to
screen the inhibitory and/or stimulatory properties of the complex wastes
before an expensive chemical analysis regime is initiated. This approach is
useful because the bioreactive components of the waste will be identified.
Those wastes which are inhibitory would be analyzed for their toxic compo-
nents, while those that stimulate productivity would not.
This approach is beneficial for at least two reasons: (1) It eliminates
unnecessary expense of organic and/or heavy metal analysis; and (2) the assay
results are usually obtained before the chemical data are available for
evaluation.
Twenty-three textile waste samples, representative of eight manufacturing
processes, were evaluated by seven assay techniques to define their toxic
properties. The bioassessment organisms included freshwater and marine algae,
crustacae, fish and mammals (Rawlings, 1978). A comparison of the sensitivity
of these bioassays (Table 12) showed that the AA:BT, using S. capricornutum,
was the most sensitive test used in the textile waste survey. This test not
only identified the toxic wastes, it also identified those that were stimu-
latory.
Forty-three percent (10 of 23) of the wastes surveyed were inhibitory and
the remaining 57% wastes were stimulatory (Shiroyama et al. in preparation).
Chemical analysis of the organic and heavy metal content of these textile
wastes was initiated at the time of collection. A savings of $19,500 could
have been realized if the AA:BT had been used to screen the wastes prior to
62
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TABLE 12
COMPARATIVE BIOTEST RESPONSES FOR TEXTILE EFFLUENTS*t
Freshwater ecology series
Textile
Plant
A
B
C
D
E
F
G
H
J
K
L
M
N
Pa
R
S
T
U
V
W
X
Y
Z
Fathead
minnow
(96-hr LC50),
% secondary
effluent
19.0
NATb
46.5
NAT
NAT
NAT
64.7
c
NAT
NAT
23.5
NAT
48.8
NAT
16.5
NAT
46.5
NAT
36.0
55.2
NAT
NAT
NAT
Daphnia Selenastmm
(48-hr EC50), (14-day EC50),
% secondary % secondary
effluent effluent
9.0 11.3
NAT
41.0
NAT
7.8 < 2.0
81.7
62.4
40% dead at 100% 7.8
concentration
NAT
NAT
28.0 12.0
60.0
100% dead at all < 2.0
dilutions
NAT
8.0 8.8
NSAd
NAT
12.1
9.4
6.3 1.0
NAT
NAT
42.6 15.5
Recommended
interpretation
Selenastrum
20% secondary
effluent
%T ] "/ ^
1 1 (4 | /o o m
53
83
187
100
95e -—
598
390
92
76
57
81
149
95e
38
95
382
1911
377
232
95
163
261
84
Marine ecology series
Sheepshead
minnow
(96-hr LC50),
% secondary
effluent
62.0
NAT
69.5
f
NAT
NAT
NAT
f
f
NAT
NAT
f
47.5
f
f
NAT
68.0
NAT
f
37.5
NAT
f
f
Grass
shrimp
(96-hr LC50),
% secondary
effluent
21.2
NAT
12.8
f
NAT
NAT
NAT
f
f
NAT
NAT
f
26.3
f
f
NAT
34.5
NAT
f
19.6
NAT
f
f
Algae
(96-hr EC50),
% secondary
effluent
f
g
90
f
10 to 50
85
59
f
f
77
1.7
f
2.3
9.0
f
9
70
g
94
50
9
f
f
Sample inadvertently collected prior to settling pond. No acute toxicity. Diseased batch of fish nullified
this analysis. No statistical analysis because heavy solids concentration obscured the analysis; the sample
did not appear to be acutely toxic. 95% growth inhibition in 27, solution of secondary effluent. Analysis not
performed on this sample. ''Growth inhibition ' 50%,in 100% solution of secondary effluent. No chemical
mutagen was detected by the 10 microbial strains. No rat mortality after 14 days due to maximum dosage of 10"5
m3/kg body weight (LD50). However, six samples (B, C, F, L, N, and S) showed potential body weight effects,
and sample R resulted in eye irritation.
63
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chemical analysis. This savings is based on the $1500 cost per analysis spent
for each of the thirteen stimulatory wastes.
64
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LITERATURE CITED
Barber, R. T. 1973. Organic "Mgands and phytoplankton growth in nutrient
rich seawater. p. 321-338 Jji P. C. Singer, [ed.], Trace metals and
metal-organic interactions in natural waters. Ann Arbor Sci.
Draper, N. R., and H. Smith. 1968. Applied regression analysis. John Wiley
and Sons, Inc., New York. 407 p.
Eppley, R. W., and W. H. Thomas. 1969. Comparison of half-saturation con-
stants for growth and nitrate uptake of marine phytoplankton. J. Phycol.
5:375-379.
Fitzgerald, G. P. 1972. Bioassay analysis of nutrient availability, p. 147-
165. Jji H. E. Allen and J. R. Kramer [eds.], Nutrients in natural
waters, John Wiley and Sons, Inc., New York.
Gerhold, R. M. 1976. Algal nutritional bioassay of Lake Wylie, North Caro-
lina, p. 175-220 In E. J. Middlebrooks, D. H. Falkenborg, and T. E.
Maloney [eds.], Biostimulation and nutrient assessment. Ann Arbor Sci.
Greene, J. C., W. E. Miller, T. Shiroyama, and T. E. Maloney. 1975. Utili-
zation of algal assays to assess the effects of municipal, industrial
and agricultural wastewater effluents upon phytoplankton production in
the Snake River System. Water Air Soil Pol It. 4:415-434.
, R. A. Soltero, W. E. Miller, A. F. Gasperino, and T. Shiroyama.
1976. The relationship of laboratory algal assays to measurements of
indigenous phytoplankton in Long Lake, Washington, p. 93-126 Jji E. J.
Middlebrooks, D. H. Falkenborg, and T. E. Maloney [eds], Biostimulation
and nutrient assessment. Ann Arbor Sci.
, W. E. Miller, T. Shiroyama, R. A. Soltero, and K. Putnam.
1978. Use of laboratory cultures of Selenastrum, Anabaena and the in-
digenous isolate Sphaerocystis to predict effects of nutrient and zinc
interactions upon phytoplankton growth in Long Lake, Washington. Mitt.
Int. Ver. Limnol. 21:372-384.
Golterman, H. L., C. C. Bakels, and J. Jakobs-Mogelin. 1969. Availability of
mud phosphate for the growth of algae. Verh. Int. Ver. Limnol. 17:467-
479.
Hall, R. H. 1973. An algal toxicity test used in the safety assessment of
detergent compounds. Presented at 36th Annual ASLO meeting, 12 June
1973, Salt Lake City, Utah.
Kuhl, A. H. L. 1964. Handling and culturing of Chlorella. Methods Cell
Physiol. 1:159-187.
Miller, W. E., T. E. Maloney, and J. C. Greene. 1974. Algal productivity in
49 lake waters as determined by algal assays. Water Res. 8:667-679.
65
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J. C, Greene, and T. Shiroyama. 1976a. Application of algal
assays to define the effects of wastewater effluents upon algal growth in
multiple use river systems, p. 77-92 Iji E. J. Middlebrooks, D. H. Fal-
kenborg, and T. E. Maloney [eds.], Biostimulation and nutrient assess-
ment. Ann Arbor Sci.
, , and . I976b. Use of algal assays to
define trace-element limitation and heavy metal toxicity. Proc. Symp.
Terr. Aquat. Ecol. Studies of NW, EWSC Press, Cheney, Wash. 1976:317-325.
Natrella, M. G. 1968. Experimental statistics. National Bureau of Standards
Handbook 91, U.S. Government Printing Office, Washington, D.C.
Odum, E. P. 1971. Fundamentals of Ecology. Third edition. W. B. Saunders
Company. 574 pp.
Payne, A. G. 1976. Application of the algal assay procedure in biostimu-
lation and toxicity testing, p. 3-28 In E. J. Middlebrooks, D. H. Fal-
kenborg, and T. E. Maloney [eds.], Biostimulation and nutrient assess-
ment. Ann Arbor Sci.
, and R. H. Hall. 1978. Application of algal assays in the
environmental evaluation of new detergent materials. Mitt. Int. Ver.
Limnol. In press.
Proctor and Gamble. 1976. Communication to Mr. S. Davis, USEPA, November
1976. Including Michigan DNR Staff report to Michigan Water Quality
Commission, August 1976; and laundry detergent usage projections based on
1973 Kline Report.
Rawlings, G. D. 1978. Source assessment: Textile plant wastewater toxics
study. Phase I. Environmental Protection Technology Series EPA-600/2-7-
004h. Washington, D.C. 153 p.
Rhee, G^Y. 1972.- Competition between an alga and an aquatic bacterium for
phosphate. Limnol. Oceanogr. 17:505-514.
Rodhe, W. 1978. Algae in culture and nature. Mitt. Int. Ver. Limnol. In
press.
Shiroyama, T., W. E. Miller, and J. C. Greene. 1975. The effect of nitrogen
and phosphorus on the growth of Selenastrum capricornutum Printz. p.
132-142 In Proc. Biostim. Nutr. Assess. Workshop, 16-17 October 1973.
U.S. Environmental Protection Agency, Corvallis, Oregon. EPA-606/3-75-
034.
T., E. A. Merwin, J. C. Greene, W. E. Miller, A. A. Leischman, and
M. A. Long. 1978. The comparative results of the AAP:BT to other
bioassay procedures in the determination of stimulatory/inhibitory
effects of textile waterwaste effluents. In preparation.
66
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Skulberg, R. 1976. Culture collection of algae. Norwegian Institute for
Water Research (NIWR) Q91, Blindern, Norway, 9 p.
Swift, D. G., and W. R. Taylor. 1974. Growth of vitamin B12-limited cul-
tures: Thalassiosira pseudonana, Monochrysis lutheri and Isochrysis
galvana. J. Phycol. 10:385-391.
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organic compounds in natural waters, p. 303-320 lr\ P. C. Singer [ed. ],
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U.S. Environmental Protection Agency, 1971. Algal Assay Procedure:Bottle
Test. Corvallis, Oregon, 82 p.
67
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11.0 APPENDICES
11.1 Bib!iography--The following references cite research using Selen-
astrum species to study the effects of nutrients, toxicants, complex wastes,
and specific inorganic and organic compounds upon algal productivity. These
citations are indicative of the importance of algal assays in the study and
management of water quality problems. Reprints of omitted or current research
citations should be sent to Mr. William E. Miller (address on title page) for
publication in future addenda. Because of Xerox regulations, reprints other
than our own are not available from the Corvallis Environmental Research
Laboratory.
Aronson, J. G., and G. L. Hergenrader. 1974. The effect of some common in-
secticides upon carbon-14 uptake in phytoplankton. Proc. Nebr. Acad.
Sci. Affil. Soc. 84:7.
Bartlett, L., F. W. Rabe, and W. H. Funk. 1974. Effects of copper, zinc, and
cadmium on Selenastrum capricornutum. Water Res. 8:179-185.
Bentley, R. E., K. S. Buxton, and B. H. Sleight, III. 1975. Acute toxicity
of five munitions compounds to aquatic organisms interim report. U.S.
Army Medical Research and Development Command - Bionomics, E.G.& G., Inc.
Contract No. DAMD-17-74-R-4755, Draft. 36 p.
Bharati, S. G., and S. P. Hosmani. 1973. Freshwater algae of Mysore State,
Part II: Chlorococcales and diatoms. Indian Sci. Cong. Assoc. Proc.
60:285.
Bilcea, R. 1975. Contributions to the knowledge of the influence of nu-
trition on the growth and development of the alga Selenastrum gracile.
Reinch. Rev. Roum. Biol. 20(3):185-191.
Bishop, N. F., M. Frick, and L. W. Jones. 1975. Photohydrogen production in
normal and mutant forms of various green algae: The requirement for
photosystem II. Plant Physiol. Supp. 56(2), p. 9.
Brezonik, P. L., F. X. Browne, and J. L. Fox. 1975. Application of ATP to
plankton biomass and bioassay studies. Water Res. 9:155-162.
Brown, E. J., and R. F. Harris. 1978. Kinetics of phosphate uptake and
aquatic microorganisms: Deviations from a simple Michaelis-Menton
equation. Limnol. Oceanogr. 23:26-34.
68
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, and . 1978. Kinetics of algal transient phosphate
uptake and the cell quota concept. Limnol. Oceanogr. 23:35-40.
Cain, J. R., and F. R. Trainor. 1973. A bioassay compromise. Phycocologia,
12(3-4):227-231.
Camp, F. A., J. M. Dolan III, and A. C. Hendricks. 1974. Algal bioassay
studies of the effects of bio-deqradation on the toxicity of a non-ionic
surfactant to Selenastrum capricornutum, Chlorophyceae. Assoc. SE Biol.
Bull. 21(2):45.
Chiaudani, G., and M. Vighi. 1974. The N:P ratio and tests with Selenastrum
to predict eutrophication in lakes. Water Res. 8:1063-1069.
, and M. Vighi. 1974. Dynamic of nutrient limitations in six
small Italian lakes, p. 28 Jji K. E. Marshall [ed.], XIX Cong. Internat.
Assoc. Limnol. 22-29 August 1974, Winnipeg, Manitoba, Canada.
, and . 1975. Dynamic of nutrient limitation in six
small lakes. Verh. Int. Ver. Limnol. 19:1319-1324.
, and . 1976. Comparison of different techniques for
detecting limiting or surplus nitrogen in batch cultures of Selenastrum
capricornutum. Water Res. 10:725-729.
, and . 1978. The use of Selenastrum capricornutum
batch cultures in toxicity studies. Mitt. Int. Ver. Limnol. In press.
Claesson, A. 1973. Algal assay procedure: Minitest with lake water (pre-
liminary report), p. 35-40 In, Algal assays in water pollution research.
Proc. Nordic Symp. , 25-26 October 1972, Oslo, Norway. NORDFORSK, Sec-
retariat of Environmental Sciences.
1978. Research on recovery of polluted lakes: Algal growth
potential and the availability of limiting nutrients. ACTA Univ. Ups.
Abstr. Upps. Diss. Soc. No. 461, 27 p.
, and A. Forsberg. 1978. Algal assay procedure with one or five
species: Minitest. Mitt. Int. Ver. Limnol. 20:21-30.
Clesceri, N. L., G. C. McDonald, I. S. Kumar, and W. J. Green. 1973. Organic
nutrient factors effecting algal growths. Ecol. Res. Series. EPA-660/3-
73-003.
Condit, R. J. 1972. Phosphorus and algal growth in the Spokane River.
Northwest Sci. 46(3):177-189.
Cowen, W. F. , and G. F. Lee. 1976. Phosphorus availability in particulate
materials transported by urban runoff. J. Water Pollut. Control Fed.
48(3)580-591.
69
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Czygan, F. C. 1970. Studies on the importance of the biosynthesis of second-
ary carotenoids as a taxanomic character in green algae. Arch. Micro-
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11.2 Recommended equipment and supplies--The AA:BT is an economical test
which can provide information not attainable by any other method. An average
cost of analysis for a comprehensive study, e.g., basic evaluation of a com-
plex waste effluent, is approximately $400.00. This consists of the evalua-
tion of four waste concentrations compared to the control receiving water,
comprising a total of five tests x three replicates or fifteen test flasks.
This cost includes basic chemical anlaysis for TSIN and Ortho-P as well as
capitalization and operation expenses. In general each test, consisting of
three replicate flasks, costs approximately $80.00. As mentioned earlier, the
AA:BT should be used to screen pollutants before establishing extensive phys-
ical, chemical and biological monitoring programs. The savings obtained will
more than pay for the initial cost of $15,000 to establish the capability to
adequately perform routine assay analysis. The following is a cost breakdown
of the necessary supplies and equipment:
1. FLASKS, Erlenmeyer, narrow mouth, heavy-duty top. (KIMAX, PYREX)*
500 ml 36/case @$37.00 (1978)
2. FLASKS, Erlenmeyer, wide mouth. (KIMEX, PYREX)
125 ml 48/case @$44.00 (1978)
3. FOAM TUBE PLUGS—Gaymar IDENTI-PLUGS. (Vendor: VWR)
200 ct @$22.00 (1978)
Order by number L800-C, size fits opening 27 to 34 mm
Manufactured by GAYMAR INDUSTRIES, INC., ONE BANK ST.,
ORCHARD PARK, NY 14127
(Cheaper foam plugs are avai 1 able—you may run into
toxicity problem with the cheaper ones; therefore check
for toxicity before using them.)
4. LIGHTS, (3) 40 w. fluorescent fixtures with (6) "cool white"
lamps and light bank frame @$85.00 (1978)
5. GYROTORY SHAKER, w/o platform. (LAB-LINE, NEW BRUNSWICK
SCIENTIFIC) @$950.00 (1978)
(better to make your own platforms, see
schematic Figure 4.)
80
-------
SHAKER PLATFORM SCHEMATIC
Req. L ist of Materials
3/4" A-C Exterior Plywood
1 20 3/4" x 45 I/4" slotted on 4 3/32" centers, slots 3/I6"
deep, 3/l6"wide, to give 4" x 4" interior dimension
compartment; 4 slots lengthwise, 10 slots crosswise, to
yield 55 compartments.
4
10
2
2
3/16" Masonite or Equivalent
3/4" x 45 1/4" strip -»
3/4" x 20 3/4" strip/ g'U6d JP
I" x 2" (Nominal) Clear Fir or Equivalent
22" strip, mitered ends
46 3/4" strip, mitered ends
Finish with 2 coats flat white paint (latex exterior
house paint acceptable).
.f
©
©
©
©
Figure 4 Schematic and list of materials for 500 ml Erlenmeyer flask
shaker platform.
81
-------
6. TEST TUBE RACK OR SUPPORT, vinyl coated. Holds 40 tubes
(5/8" diameter and 7 7/8" L x 3 1/2" W x 3 1/4"
- @$8.00 (1978)
H)
7. BELLCO BEAKER, Modified for coulter counter (BELLCO)
12/box @$25.00 (1978)
BELLCO GLASS, INC.
340 EDRUDO RD
VINELAND', NJ 08380 Tel: 609/691-1075
8. MICRO PIPETTING SYSTEM. 1 ml w/o tips. (OXFORD,
EPPENDORF) @$49.00- (1978
$65.00
Disposable tips 1000/pk -- @$45.00- (1978)
$55.00
9. PIPETTOR. 1.0 to 10.0 ml dispenser. (OXFORD, REPIPET,
UNIVERSAL) @$80.00- (1978)
$160.00
10. MILLIPORE MEMBRANE
A. 0.45 urn, 47 mm diameter plain, autoclaved
pack or sterile pack 100/pk @$24.00 (1977)
0.22 [jm, 47 mm diameter, plain, sterile
100/pk @$24.00 (1977)
B. Millipore funnel hydrosol stainless 47 mm- @$186.30 (1977)
OR
PYREX 47 mm Glass Funnel @$44.80 (1977)
Teflon-faced Pyrex 47 mm funnel @$55.00 (1977)
MILLIPORE CORP.
BEDFORD, MASS 01730 Tel: 800/225-1380,
in Mass., (617)275-9200
11. COULTER COUNTER ZBI, w/ 70 & 100 u aperature tube @$8000.00 (1978)
MCV/HCT Flatpack to go with ZBI —— @$3500.00 (1978)
COULTER ELECTRONICS, INC.
590 WEST 20TH ST
HIALEAH, FL 33010
12. HEAT EXCHANGER ---cost depends on room size and
number of light banks and shakers as well as
ability of facility to maintain temperature
within 20°C.
* Mention of Trade names or commercial products and sources does not consti-
tute endorsement by the U.S. Environmental Protection Agency.
82
-------
11.3 FORTRAN data reduction program—The algal assay data analysis
system consists of two programs: (1) ALGASSY (pages 89-94), which reads the
data cards and produces a line printer summary and a data file which is input
to; (2) ASSYPLOT (pages 95-98), which produces plots of mean dry weight versus
time. These programs written in FORTRAN IV are currently running on the CDC
3300 operated by the Miline-Computer Center, Oregon State University. List-
ings of these programs and sample input and output are provided for analysis
and use in establishing a similar data reduction format.
Completed data reduction formats for assays conducted on a test water
collected from Long Lake, Washington (pages 84-88) are included as examples of
data reduction used to facilitate computer enumeration and plotting of assay
data. Note: Line 0010 in ALGASSY program (page 89) is the inclusion of the
older dry weight yield conversion factor (2.0 x 10-7) as determined in section
8.4. This factor is now 3.6 x 10-7 and may differ according to values ob-
tained by each investigator.
83
-------
ALGAL GROWTH POTENTIAL TEST
ALGAL ASSAY TEST CODE: LBOV Z3 77
MEDIA: /.ova 4»*«j wa. VOLUME, flask:
PRETREATMENT:
TEST ORIGINATOR:
TEST ORGANISM:
RESPONSIBLE TECHNICIAN:_
„! solution: /00 m>
INOCULUM SIZE: /0ao .„,. m/.
DATE: to-7-71
SPIKE: UNINOCULATED CONTROL fUNC)
PHOSPHORUS (P) .os EDTA (E) y.0 OTHER
COMPLETION DATE: /o-aa-77
CONTROL (C) ^ NITROGEN (N) ,.0
fl+r.
CHEMICAL ANALYSIS REQUIRED: AAM-F .
METAt. -F t
COUNTING DAYS: 1 ,2,3,4 ,5 ,6 0*8,9,10 dJU 2,13 45J1 5,16,17,18,19,20,21.
COLLECTION FLASK
DATE NUMBERS
¥-2.3-77
CHEMISTRY pH pH
LAB. CODE ORIGINAL PRETREATED
7.Q7 7.ot
1- 3 C.«-Ar0l
4- 6 /.o~a AJ /-'
7- 9 c>.oj^ P ;-'
10-12 A/*J»
13-15 /.O«5 JF2>TA /-'
16-18 A/-*-£
19-21 p + E
OO O /I
cc-c.^ f\l t P 4- £"
25-27
28-30
31-33
34-36
37-39
40-42
43-45
46-48
49-51
52-54
55-57
58-60
61-63
64-66
67-69
70-72
73-75
76-78
79-81
82-84
85-87
88-90
91-93
94-96
97-99
100-102
103-105
106-108
109-111
112-114
115-117
118-120
121-123
124-126
127-129
130-132
133-135
136-138
139-141
142-144
145-147
148-150
151-153
154-156
157-159
160-162
163-165
166-168
169-172
173-175
176-178
178-181
182-184
185-187
188-190
NO ItS: -
Figure 5. Completed algal growth potential test design format.
84
-------
Date Sampled Apt-// Z3, 1*177
TEST CODE: LB
MEDIA: AF Aou^ /.?*e , Wa .
SPIKE: C, /.O^A//-\ o QJ^ P/-' /.Qm
A/*/* >
SPIKE RANGE:
TEST VOLUME: _ /oo mf
CONTAINER VOLUME:
# REPLICATE FLASKS:
STOCK CULTURE DATA: INOCULUM:
MEDIA: /oo% A A M Sa/gA/»5^rM.i~v Alga
DAYS GROWTH: 7 _L°_°+. ___ cells/nl
Q. o// __ rnq/1 Pry V/t.
MCV
TEST PREPARATION:
TEST WATERS:
DISPENSED:
ML
ML
SPIKED: ML
INOCULUM: ML
INOCULATED:
SAMPLED:
COUNTED:
ML
ML
ML
FRESH SPIKE:
Figure 6. Completed growth assessment cover sheet
85
-------
LAB*
tn
_l
6
ur
_Z
3
flt
JL
L±l
N
t
L*J
i
i
0
TREATMENT
L
-V
9
SPIKE
f]
u,
SPECIES
LS
1415
clu
INOC.,
MG./L
7
o
IP
19
i
1
CELL COUNTS
MCV C MCV
LAB
TREATMENT
DAYIDH.FACT.Iniaipn ,R
[P"-^CT.I|
lr.V4iS ?,j'
'lM-4>ahl7^'7l2{J29J43
07
2I°I I
MM^ |2'°' l3
15
18 .
CELL COUNTS
MCV C MCV
3 £
LAB* 4J
nqq
AF
541 535Cu7l&8
61 62 53 it 6
SPECIES
MCV
66 57
OAYIDIL.FACT.
117
HL.FACT.I^KQgfl
7'4p |2{T7 2f|?
zi^rsij
"3
l/l/l I l^lol lauri
^
I l*lol
TREATMENT
C
SPIKE
INOC.,
MG./L .
11
jCZ
CELL COUNTS
MCV C MCV
5152.
54
f^.
^.*
TREATMENT
[DAYIDIL.FACT.lEKBPn 'R I C
ULL
SPIKE
MCV
66 37 >ffl
L3.1
SPECIES
sIcLk
107
'5 ?pb?l2329 "3
^*" ™*V^^^~^«"" •*»
2. O
3
l/lvl
15i4d47J8i9
CELL COUNTS
MCV C MCV
INOC.,
MG./L .
ra
!6n
31 625314 6
C
Figure 7a. Completed growth assessment data sheet.
86
£
MCV
5Ztf
-------
EXPERIMENT I.D.-J*J££±£1ZZ_ LOCATION '-"ft L'
• w'-
AF
SPECIES
TREA TMENT
SPIKE
n
s
v
E
15
j.
INOC.,
MG./L .
17
0
IF
I
19
I
!Qi
1
•LLL
CELL COUNTS
MCV C MCV
SI
5££Z
-f
4 J
6?
63
&£
MCV
S657
AF
TREATMENT
IDAYIDIL.FACT.lGKflpn )R C
inh
SPIKE
L3
SPECIES
1415
IE
07
i*z
Zioiol
} 43
io[c
jjj-l
45|4647h8i9
016
LO\&
M
CELL COUNTS
MCV C MCV
3 /
AisascU
£
s.s.
58 5a
r
4m
041
INOC.,
MG./L ..
MCV
61 6253'
/\0
6^7
18
TREATMENT
CELL COUNTS
|DAY|DIL.FACT.|ctr,pn IR | C | MCV C MCV
o|7l I2lol
%
I'"* I
izjo o
|2
15
19
2jo
4 SISCu
70
7 Z
Figure 7b. Completed growth assessment data s
2C
Z.J.
MCV
iS?V
neet.
87
-------
EXPERIMENT IDENTIFICATION
ALGAL ASSAY
EPA
FORM
This form is to identify to the computer the nature of the experiment to be processed
The information contained herein is to be keypunched; please print clearly.
Experiment Name Predicted Yield Sampling Site Description
oo
OD
A/
1 10 17
Figure 8. Completed Computer ID format.
-------
0001
0002
0003
0004
0005
0006
0007
oooa
0009
0010
0011
0012
0013
0014
0015
0016
0017
0013
0019
0020
0021
0022
0023
0024
0025
0026
0027
0028
0029
0030
0031
0032
0033
0034
0035
0036
0037
0038
0039
0040
0041
0042
0043
0044
0045
0046
0047
0048
0049
0050
0051
0052
0053
0054
0055
<
<
4
2001
2002
2003
2004
2006
2007
200fl
201?
2013
1001
1002
1003
1007
1010
PROGRAM ALGASSY
INTEGER ALG.OAY
COMMON OUTPUT(60,10).CODE(10).NUMSPIKE.OLOLAB,YI ELD.EXPERMNT.
SITE(4).CCAVG(60),CCSV(60).DWAVG(60) ,DWSV(60) ,CN(60).I TOLD.
TRTNAME(3.10).ORGANISM(2.10).IORG.DWI
DIMENSION RKGO(5).C(3).DW(3).CC(3).Y(10).KODE(IO),
CV(3),SPTKODE(20).ERROR(3).OAT A(9)
DATA(ERROR="SPTKE "."TRTMENT "."SPECIES ")
OATA(KODE="SEL")
DATA(Y=2.0E-7)
DATA(ORGANISM="SELENAST","RUM ")
OATA(TRTNAMF="AUTOCLAV"."FD AND F"."ILTERED "."AUTOCLAV".
• "ED ONLY "," "."FILTERED"." AND AUT"."OCLAVED ".
"FILTERED"." ONLY >'t" ")
OATA(SPIKOOE="CONTROL "."1.0 N ","N»P ",
"1.0 E ","N*E "»"P»E "."N+P*1,0£"»
• "UNINOC "."0.05 P ")
FORMAT(4X,A3,F5.4,I2,F4,5F3,I2,X,3(F5,F3))
FORMAT(///,5X."EXP:".A8." LAR:".A7»X.3A8,X.A8.X.2A8.X,4A8»/»
• " DAY DRY WEIGHTS MEAN STD T",
* 11X."CORRECTED COUNTS".9X."MEAN".7X,"STD".7X,"T"»/)
FORMATU6.6F8.3.5F10.F8.3)
FORMAT(A2)
FORMATdHl)
FORMAT(9A8)
FORMAT(A7»2I2)
FORMAT(" LAB ".A7."» BAD ".A8)
FORMAT<" LAB ",A7." DAY LT 1 OR GT 60")
CCF=.000002
MAXSPIKE=19
MAXTRT=9
MAXORG=10
FIOST=PAGE=0.
IDONF=NUMDAYS=0
ONPAGE=3.
EXPFRMNTsflH
REAO(1.2007)DATA
IF(.NOT.EOFd) )GO TO 1002
CALL OUT11
STOP
DECOHE(2,2004,DATA(1))IP
IF(IP.EQ."63".OR.IP.EQ." ".OR.IP.EQ."56")GO TO 1010
TTEST="NO "
IF(DATAd) .NE.EXPERMNT)PAGE = 0.
IF(FIRST.FO.O.)GO TO 1003
CALL OUT11
FIRST=1.
ITOLO=ISPOLD=NUMSPIKE=0
DO 1007 1=1,4
SITEd)=DATA(I»2)
CONTINUE
YIELD=DATA(2)
fXPERMNT=DATA(l)
GO TO 1001
DECODE(11,2008,DATA(l))FLAB,IT,ISP
Figure 9a. ALGASSY, computer program data reduction printout.
89
-------
06/01/78
3:14 PM
ATEMP
PAGE
0056
0057
005fl
0059
0060
0061
0062
0063
0064
0065
0066
0067
0068
0069
0070
0071
0072
0073
0074
0075
0076
0077
0078
0079
0080
0081
0082
0083
0084
0085
0086
0087
0088
0089
0090
0091
0092
0093
0094
0095
0096
0097
0098
0099
0100
0101
0102
0103
0104
0105
0106
0107
0108
0109
0110
1011
IFGO TO 1013
WRITF(61,2012)FLAB«ERROR(1)
GO TO 1001
DECODE (60. 2001 .DATA (2) ) ALG f OWI f DAY f D.RKGD. I REP.
* (C(D »CV(I) .1=1.3)
DO 1014 IORG=1.MAXORG
IF(ALG.EQ.KODEUORG) )GO TO
1014 CONTINUE
WRITE (61, 2012) FLAB. ERROR (31
GO TO 1001
1015 IFIDAY.GE. LAND. DAY. LE. 60)60 TO 1016
WRITF(61,2013)FLAB
GO TO 1001
C DATA HAS PASSFO CHECKS
1016 IF(ISPOLD.EQ.ISP)GO TO 1020
NUMSPIKE=NUMSPIKE»1
CODE (NUMSP IKE> =SPIKOOE ( ISP)
ISPOLD=ISP
IF ( P AGE. FQ.O.) WRITE (3 1.2006)
WRITF(31,2002>EXPERMNT,FLAB, ( TRTNAMF ( I ; I T) » 1=1.3) .SPIKODE < ISP) »
« (OPGANISM(I,IORG) « 1=1.2) .SITE
IF(inONE.NE.l)(50 TO 1019
IF(NUMDAY«5.LE.9)ONPAGE = 4.
1019 IOONE=IDONE»1
PAGF=PAGE*I .
IFPAGE=O.
1020 TiMFS=SUM=o.
DO 1021 1=1,5
IF(BKGOd) .EO,0.)GO TO 1021
SUM = SUM»BKr,DU)
TIMES=TIMES*1.
CONTINUE
IX=IFIX(SUM/TIMES+.5)
X=FLOAT(IX)
CALCULATE D»Y WEIGHT AND CORRECTED COUNTS
1021
DO 1022 1=1,3
IF(C(I) ,EO.O.)GO TO 1022
COUNTS=COUNTS»1.
CSUM=CSUM*C(I)
CVSUM=CVSUM»CV(I)
102? CONTINUE
IF(CnUNTS.LF..O.)GO TO
AVGC=CSUM/COUNTS
AVGCV=CVSUM/COUNTS
DW(IOEP)=.0001
1023
Figure 9a
90
-------
06/01/78
3:14 PM
ATFMP
PAGF
0111
Oil?
0113
0114
011S
0116
0117
Ollfl
0110
0120
0121
0122
0123
0124
0125
0126
0127
012ft
0129
0130
0131
0132
0133
0134
0135
0136
0137
0138
0139
0140
0141
0142
0143
0144
0145
0146
0147
0148
0149
0150
0151
0152
0153
0154
0155
0156
0157
0158
0159
0160
0161
0162
0163
0164
0165
CCIFF°(l . »CCF»DIFF ) « AVGCV
CC(IPEP>=0«OIFFM1..»CCF<>DTFF)
1023 IF(IREP.LT.3)GO TO 1001
c CALCULATE MFAN AND STANDARD DEVIATION
CCSUM=CCSUMSO=DWSUM=DWSUMSQ=REPS=0.
00 1024 1=1,3
IF(DWU) ,NE.O.)REPS = REPS*1.
DWSUM=DWSUM+nw < I »
DWSUMSO=DWSUMSQ»OW(I)«DW
CCSUMSQ = CCSUMSQ»CCm«CC(I)
1024 CONTINUE
DWMEAN=DWSUM/REPS
OUTPUT(DAY.NUMSPIKE) =DWMEAN
CCMEAN=CC5UM/REPS
OWSTD=CC5TO=0.
IF(PFPS.LT.2.)GO TO 1025
SVOW= (DWSUMSQ-DWSUM»DWSUM/REPS) / (REPS-1. »
OWSTn=SQRT(SVQW)
SVCC= (CCSUMSQ-CCSL)M«CCSUM/REPS) / (REPS-1 . )
CCSTO=SQRT (SVCC)
1025 TCC=TDW=0.
IF(I«5P.NE.1)GO TO 1026
IF(RrPS.LT.2)GO TO 1026
TTE5T="YES "
C SAVE CONTROL DATA FOR T TEST
OWAVG(OAY) =DWM£AN
DWSV(DAY)=SVD«
CCAVr,(DAY) =CCMEAN
CCSV(DAY)=SVCC
CN(OAY)=REPS
GO TO 1027
C COMPUTE T STATISTIC
1026 IF(TTEST.EO."NO »)GO TO 1027
IFtCN(DAY) .LT.2..0R.OEPS.LT.2.)GO TO 1027
DOw2=(CN(OAY)*REPS)»<(CN(r>AY>-l.)°nWSVOAY,DW,OWMFAN,nwSTD,TO*«CC»CCMtAN,CCSTD,TCC
IF ( IOONE.F0.1)NUMQAYS = NUMDAYS*1
DO 1028 1=1,3
OW(I)=CC(I)=0.
CONTINUE
GO TO 1001
FND
SUBROUTINE OUT11
COMMON OUTPUT(60,10) ,CODE(10) ,NUMSPIKE,FLAB»YI ELD,EXPEPMNT»
• SITE(4),CCAVG(60),CCSV(60),DWAVG(60),DWSV(60),CN(60),IT,
Figure 9a
91
-------
06/01/78
3:14 PM
ATEMP
PAGE
0166
0167
016R
0169
0170
0171
0172
0173
017*
0175
0176
0177
0178
0179
01BO
0181
0182
0183
0184
0185
0186
0187
0188
0189
0190
0191
0192
0193
e
2101 i
2102 I
2103 1
2104 i
«
1101
1102
1103
1104
1105
1106
TRTNAMF(3tio>»ORGANISM;?.10).IORG.DWI
<12X.10A8)
FORMAT(8X,T4,10F8.3)
FORMAT(A8.3X.5A8)
FORMAT(8X.A7,X.3A8.X»2A8.F8.4)
W»ITF(11,2103)FXPFRMNT, SITE.YIELD
WRTTF(ll,2104)FLAH,(TRTNAME
-------
EXP;LB04?377 LAB.-6342023 AUTOCLAVED AND FILTERED CONTROL SELENASTRUM LONG LAKE. WA L101477B
DAY DRV WEIGHTS MEAN STD T CORRECTED COUNTS MEAN STD
7
11
14
.236
.497
.503
.101
.407
.229
.268
.453
.545
.202
.453
.427
.089
.045
.173
11560
21299
22276
4809
12649
10992
11406
18681
21573
9258
17543
18280
3854
4436
6322
EXP:LB04?377 LAB:6342023 AUTOCLAVED AND FILTERED 1.0 ~N~ SELENASTRUM LGNG~LAKE. WA L1014778
DAY DRY WEIGHTS MEAN STD T CORRECTED COUNTS MEAN STD
11
14
". 25fl
.462
.490
.206
.240
.237
.280
.485
.409
.248
.395
.379
.038
.135
.129
.833
.694
.389
11781
19625
20033
9322
1C752
9576
11012
18829
16106
10705
16402
15238
1258
4909
5282
.618
.299
.640
EXP:L804?377 LAB:6342023 AUTOCLAVED~AND"FILTERED "0.05 P
DAY DRY WEIGHTS MEAN STD T
"SELENASTRUM LONG"LAKE» WA L101477B
CORRECTED COUNTS MEAN STD
T" .353 "".314" .612 .426" " .162 " 2.T01 ""16220" ""13592 29312 1970S 8421 1.954
11 .949 .683 2.182 1.271 .800 1.770 42755 28219 94340 55105 34748 1.857
14 1.696 .657 3.911 2.088 1.662 1.721 66233 26497 149291 80674 62658 1.716
EXPILB042377 LAB:6342023 AUTOCLAVED AND FILTERED N+P
DAY DRY WEIGHTS MEAN STD T
SELENASTRUM LONG LAKE, WA L1014778
CORRECTED COUNTS MEAN STD
"7
11
14
.273
.456
.596
.214
.249
.328
.176
.300
.294
.221
.335
.406
.049
.108
.166
.332
1.739
.154
11527
20495
24044
9021
11132
14936
7753
12669
120*8
9434
14766
17009
1320
5021
6261
.070
.718
.247
Figure 9b. ALGASSY, computer program reduction printout.
-------
EXP;_LB04?377_ LAB:6342023 AUTOCLAVED AND FILTERED 1.0
SELENASTRUM LONG LAKE. WA L101477B
STD
'Ar DRY WEIGHTS
7~
1)
14
20.97?
22.797
25.620
21.493
22.149
24.963
20.847
20.290
25.876
MEAN
21 . IC4
21 . 745
25.487
STD
.343
1. 3Q2
.472
102.
28.
86.
T
205
317
392
CORRECTED COUNTS
"1621541
1790355
1940875
""1628283
1730417
1891139
1663316
1618858
2032305
MLAN
1637713
1713210
1954773
22427 123.949
87034 33.702
71602 46.662
EXP:LB04?377 LAd:6342023 AUTOCLAVED AND FILTERED N+E
DPY WEIGHTS MEAN STD T
SELENASTRUM LONG LAKE* wA L101477B
CORRECTED COUNTS MEAN STD
7
11
14
" ~EXP
DAY
11
14
35.226 33
37.710 36
42.937 38
~:LBO~42377
DRY
20.758 22
22.595 24
24.405 26
.383 34.937
.150 36.699
.804 38.432
L A8: 634 20 23~
WEIGHTS
.485 20.900
.841 22.441
.522 24.724
34.515
36.853
_40 = 053
AU"TOCLAV"ED~
MEAN
" "21.381
23.292
25.217
.991
.791
2.5Q1
~AND~F
STD
~ .959
1.343
1.141
59.712
79.531
27.384
lil'TEKEDT
T
387091
29.437
37.200
2113543
2308777
2586585
P + E
1556844
1711738
1906629
1987055
2195321
2318950
SELENASTRUM
CORRECTED
1686385
1872404
1989133
2023363
2133681
2183641
LONG
COUNTS
1667516"
1781039
1941705
2041320
2212593
2363059
LAKE, VJA
MEAN
1636915
1788394
1945823
65128
88316
205061
"L1014778
STD
69982
80585
41406
53.947
42.753
19.796
T
40.223
38.004
79.707
EXP:LB04?377 LAB:6342023 AUTOCLAVED AND FILTERED
DAY DRY WEIGHTS MEAN STD 1
N"+P*C SELENASTRUM LONG LAKE. WA L101477H
CORRECTED COUNTS MEAN STD
7" 5ft.I49
11 66.717
14 70.671
57.056
63.456
71.049
"57.453
67.716
68.820
"57.553
65.963
70.180
~ .553 177.323
2.228 50.920
1.193 100.245
3926979"
4633144
4885100
"4094948
4759207
5249927
4045992
4836867
5011187
"4023306
4743073
5048738
85278 81.444
102815 79.533
185289 46.996
Figure 9b
-------
05/26/78
i:4b
ASSYHi-OT
HAGt 1
0001
0002
0003
0004
OOOb
0006
0007
oooa
0009
0010
uoii
0012
0013
0014
OOlb
0016
0017
0018
0019
0020
0021
0022
0023
0024
002b
0026
0027
0028
0029
0030
0031
0032
0033
0034
0035
0036
0037
0038
0039
0040
0041
0042
0043
0044
004b
0040
0047
0048
0049
0060
0051
00b2
0033
00b4
OObb
2001
2002
2003
2004
2uOb
200b
1001
1002
C DK,
1003
1004
»-l".
H»,
U
"0 '
it Y
i,nl i
M T
PROGRAM AbbYHLOT
DIMENSION LX(ll),LY(7),DM(10)«UATA(12> tOHGANIbM(2>
> YB<2)t FRT(3)«IbP<10)tYLAB£L(3).MKK(IO) (SITE(4)
> POINTS (20(10) tbPIKODEdO) » IN (20) » AB1 Ab (2)
OATA(XBlAb=0.»lb.)
UATA(XB=0.t24.)
DATA(YB=-3.»3.)
DATA(LX=»0 "»"4
OATA(LY = "-3'S"-
OATA(YLABEL=««M G
DATA(MRK=2f4
FORMAT(12AB)
FORMAT(3x.4AatF8.3)
FORMAT(8x,A/»x»3Aa»x»2A8»F8.4)
FORMAT(i2x«IOAB)
FORMAT(F4,10f-8.3)
FORMAT(F4," PLOTS PRODUCED")
CALL TK4010
CALL PLOTTYPt(O)
ICROSS=1
CALL SIZc(29.tl2.)
"»"Jo»»»*0
OAYLABEL = ''D A Y S"
TEN="10"
PLOTb=0.
CALL
1001 R£AD(l<200l)OATA
IFI.NOT.LOF(1))GO TO 1002
GO TO 1003
1002 IF(DATAd) .EU.8H ) GO TO 1022
!F(PLOTb.EU.O.)GO TO 1020
C DKAW HORIZONTAL BOUNDARIES
IF(DAYLAbT.LT.2<».)GO
ID=IFIX(UAYLAST«.1)
IFdO.GT. 39) 10 = 39
TO
UIM = <»» (NX-i)
XB(2)=FLOAl (LIM)
CALL SCALt(.25»1.5.XBlAS(ICROSS) .0..-6,
DO lOOb !B=lt2
CALL PLOT (U.» YB ( IB) »0»0)
00 1003 J=1»LIM
X=FLOAT (J)
MARK=7
MULT=J/H
IF ( IP.tU.O)
IF ( J.tU.L IM) MrtRK=0
CALL PL 01 (At fb( Ib) , ItMARK)
Figure 10
95
-------
05/20/7%
HM
ASSYPLOT
PAbE
0036
0037
U03B
0039
0060
0061
0062
0063
00t>*
0065
0066
0067
0068
006V
oo7o
0071
0072
0073
0074
00/6
0077
0078
0079
0080
0081
0082
0083
0084
008b
0066
0087
0088
0089
0090
0091
0092
0093
0094
009b
0096
0097
U098
0099
0100
0101
0102
0103
010*
OlOb
0106
010?
010«
0109
olio
CONTINUE
1006 CONTINUE
C LABEL X AXIS
CALL PLOT <-.2»-3. 2,0,0)
X=-.2
00 1007 I=1»NX
CALL SYMBuL
Y=.00i
00 100V IUtC=l.b
DY=Y
00 lOOb 1=1.6
1008
CALL PLOT(XBdb) ,EXP,1,MAHK)
1009 CONTINUE
1010 CONTINUE
C LABEL Y AXIS
TENY=-3.0b
EXPY=-2.9
00 1011 1=1,7
CALL SYMeUL(-2.,T£NY,0.,.l6,^,TEN)
CALL iYMbOL<-.d»hXHY,0.».0«,2,LY (I) )
TENY=TtNY»l.
EXHY=EXPY»1.
1011 CONTINUE
CALL SYMt)UL(-3.»-l.,90.,.l6.18»YLABEL)
CALL SYMt)OL(-3.4,l.lb,90.,.08»«r»LY(3> )
CALL SYMt)OL(-b.»-J.,90.,.lb,16»ORGANISM)
CALL SYMbOL <-b. »-l.»(»0.» . 16,24, TUT)
CALL SYMdOL(-5.»i;.,90.,.16»7,FLAd)
= ALU(ilO( Y)
CALL PLOT (XtMIti) ,tXP, 1,5)
CONTlNUt
Y=Y»DY
EXP=ALUG10(Y)
MAftK=6
PLOT INOCULUM CuNCtN FK A F I ON
IF(0»»I.LT..oul.OH.OwI.GE
ANO PHtOICTED YIELD
1000.)(JO TO 1012
CALL SYMBOL <.2»riNoc»u.».OB«i»io«)
1012 IF (YIELD. LT. .001 .OH. YIELD. GE. 1 UOO. ) bO TO 101J
X=X8(2)-1.
Y = ALOG1(J ( YltLO)
CALL SYMbOL (A, T » U. » .UO, 1 .
C PLOT POINTS
101J 00 lOlb I =
CALL PLOT < .3, YiNOC,o,o»
Figure 10
96
-------
05/26/78
1:45
ASSYHLUT
PAGE
0111
0112
0113
0114
0115
Ollb
0117
Olid
0119
0120
0121
0122
0123
0124
0125
0126
0127
oi2«
0129
01JO
0131
0132
0133
0134
0135
0136
0137
0138
0139
0140
0141
0142
0143
0144
00 1014 N=l,NUAr
IF(PUINfb
-------
ro
N
o
N
sr
ro
ID
Q
Ld
o:
u
h-
Q
2
-------
11.4 Standard algal assay forms—The following data reduction and exper-
imental design formats are intended as a guide to facilitate laboratory ident-
ification and data analysis for test waters which are assayed.
99
-------
Figure 11. Algal growth potential test design format
100
-------
ALGAL GROWTH POTENTIAL TEST
ALGAL ASSAY TEST CODE:
MEDIA: VOLUME, flask: solution?"
PRETREATMENT: TEST ORGANISM: INOCULUM SIZEj_
TEST ORIGINATOR: DATE:
RESPONSIBLE TECHNICIAN: COMPLETION DATE:
SPIKE: UNINOCULATED CONTROL (UNC) CONTROT (C) NITROGEN (N)
PHOSPHORUS (P) EDTA (E) OTHER "
CHEMICAL ANALYSIS REQUIRED:,
COUNTING DAYS: 1 ,2 ,3,4,5 ,6,7,8,9 ,10 ,11 ,12,1 3,14 ,1 5,16 ,1 7 ,18,19 ,20 ,21.
COLLECTION FLASK CHEMISTRY pH pH
DATE NUMBERS LAB. CODE ORIGINAL PRETREATED
1- 3
4- 6
7- 9
10-12
13-15
16-18
19-21
22-24
25-27
28-30
31-33
34-36
37-39
40-42
43-45
46-48
49-51
52-54
55-57
58-60
61-63
64-66
67-69
70-72
73-75
76-78
79-81
82-84
85-87
88-90
91-93
94-96
97-99
100-102
103-105
106-108
109-111
112-114
115-117
118-120
121-123
124-126
127-129
130-132
133-135
136-138
139-141
142-144
145-147
148-150
151-153
154-156
157-159
160-162
163-165
166-168
169-172
173-175
176-178
178-181
182-184
185-187
188-190
NOTE5
101
-------
Figure 12. Dilution test design format
102
-------
DILUTION TEST DESIGN
ALGAL ASSAY TEST CODE: CHEH. LAB. CODE_
TEST ELEMENT OR COMPOUND:
SPIKES:
MEDIA: VOLUME, flask solution_
COLLECTION OR PREPARATION DATEj PRETREATMENTj
TEST ORGANISM: INOCULUM SIZE:
COUNTING DAYS: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41.
CHEMICAL ANALYSIS REQUIRED:
TEST ORIGINATOR:
RESPONSIBLE TECHNICIAN
DATE:
COMPLETION DATE
1- 3
4- 6
7- 9
10-12
13-15
16-18
19-21
22-24
25-27
28-30
31-33
34-36
37-39
40-42
43-45
46-48
49-51
52-54
55-57
58-60
61- 63
64- 66
67- 69
70- 72
73- 75
76- 78
79- 81
82- 84
85- 87
88- 90
91- 93
94- 96
97- 99
100-102
103-105
106-108
109-111
112-114
115-117
118-120
NOTES OR SPECIAL INSTRUCTIONS:
103
-------
Figure 13. Dose/response test design format
104
-------
DOSE/RESPONSE TEST DESIGN
ALGAL ASSAY TEST CODE: __CHEM. LAB. CODE_
TEST ELEMENT OR COMPOUNDj
SPIKES:
MEDIA: VOLUME, flask solution_
COLLECTION OR PREPARATION DATEj PRETREATMENTj
TEST ORGANISM: INOCULUM SIZE:
COUNTING DAYS: 1 ,2,3,4 ,5,6,7,8,9 ,10,11 ,12 ,1 3 ,14,15 ,16 ,17 ,18,19 ,20 ,21 ,22,
23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41.
CHEMICAL ANALYSIS REQUIRED:_
TEST ORIGINATOR:
RESPONSIBLE TECHNICIAN
DATE:
COMPLETION DATE
1- 3
4- 6
7- 9
10-12
13-15
16-18
19-21
22-24
25-27
28-30
31-33
34-36
37-39
40-42
43-45
46-48
49-51
52-54
55-57
58-60
61- 63
64- 66
67- 69
70- 72
73- 75
76- 78
79- 81
82- 84
85- 87
88- 90
91- 93
94- 96
97- 99
100-102
103-105
106-108
109-111
112-114
115-117
118-120
NOTES OR SPECIAL INSTRUCTIONS:
105
-------
Figure 14. Growth assessment data cover sheet
106
-------
Date Sampled^
TEST CODE:
MEDIA:
SPIKE:
SPIKE RANGE:
TEST VOLUME:
CONTAINER VOLUME:
# REPLICATE FLASKS:
STOCK CULTURE DATA: Inoculum;
MEDIA: Alga
DAYS GROWTH: cells/ml
mq/1 Dry Wt.
INOCULATED:
SAMPLED:
COUNTED:
FRESH SPIKE:
MCV
TEST PREPARATION
TEST WATERS:
DISPENSED:
SPIKED:
INOCULM:
107
-------
Figure 15. Growth assessment data sheet (short form)
-------
LOCATION
START DATE.
FLASK
1234
LAB
NIIMRFRR
567 89
# TREAT-
MENT
loTjT 13 14
JL _
INOC.,
15 17 18
19
20
SPIKE SPECIES MG/L
DAY
21
22
DILUTION
FACTOR
23
24
25
26
BACK-
GROUND
27
28
29
E
__J
IE
R
43
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
COUNTS
45
46
47
48
49
MCV
50
51
52
COUNTS
53
54
55
56
57
MCV
58
59
60
COUNTS
61
62
63
64
65
MCV
66
67
68
CALC.DRY
WEIGHT
INOC.,
|l
L
2
3
4
5
6
7
m . ..
10
4.
13
14
15
17
18
19
20
LAB # TREAT- SPIKE SPECIES MG/L
MENT
DAY
21
22
DILUTION
FACTOR
23
24
25
26
BACK-
GROUND
27
28
29
.
R
43
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
'i
3
COUNTS
45
46
47
48
49
MCV
50
51
52
COUNTS
53
54
55
56
57
MCV
58
59
60
COUNTS
61
62
63
64
65
MCV
66
67
68
CALC. DRY
WEIGHT
109
-------
Figure 16. Growth assessment data sheet (long form),
110
-------
LOCATION
START DATE
FLASK NUK
123456
LAB #•
1PFR^
7 |8[9J 15
I I I
INOC.
Tii is i4 1
:j :
5 17 18
19
f
20
TREAT- SPIKE SPECIES MG/L
MENT
DAY
21
22
DILUTION) BACK-
FACTOR IGROUNC
23
24
25
26J27
1
28
29
__
_
R
43
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
COUNTS
45
46
47
48
49
MCV
50
51
52
COUNTS
53
54
55
56
57
MCV
58
59
60
COUNTS
61
62
63
64
65
MCV
66
67
68
CALC. DRY
WEIGHT
111
-------
Figure 17. Computer ID format
112
-------
EXPERIMENT IDENTIFICATION
ALGAL ASSAY
EPA
FORM
This form is to identify to the computer the nature of the experiment to be processed.
The information contained herein is to be keypunched; please print clearly.
Experiment Name Predicted Yield Sampling Site Description
CO
10
17
-------
Figure 18. Dry weight versus time format
114
-------
10'
o>
E
I
H
10'
10"
,-2 i i I I i i i I i i i I i i i I I I I I i I I
8
12
DAYS
16
115
-------
Figure 19. Dry weight versus any x value format
116
-------
10
10'
xlO
I
h-'
£io<
10
-I
10
117
-------
Figure 20. Cell counts versus days format
118
-------
io7
10*
UJ
U
io5
io4
10'
I ' ' ' I
-I—J 1 ' ' ' I ' ' ' I ' I L
04 8 12 16 20 24
DAYS
119
-------
Figure 21. Dry weight versus cell counts format
120
-------
ro
10
Iio6
LJ
O
10"
10
Mill I T I I I |ll| I I I I Mill I I I I INN I I I I HIM I I I I Illl
3 i i 1111ni i i 11111il i 1111 iiil i i 11 mil i i i 11 ml i i 11 mi
.0!
10 100
DRY WT. — Mg/L
1000
-------
11.5 MCV calibration—The threshold must be set on the Coulter Counter
and on the MCV Computer, and the Computer must be calibrated before analyzing
samples. The calibration adjustments need not be repeated more often than
about once a week unless the Coulter Counter control settings have been dis-
turbed for other types of samples. The MCV Computer calibration should be
checked every day or at the beginning of each work shift to insure that the
results are accurate.
A dilution made by adding one drop of well mixed Organic Calibration
Material Lot #13020 to about 20 ml of Isoton II, or 1% NaCl, is required for
the threshold adjustments with a lOOum aperture tube, and for the Computer
calibration and operation check.
Turn the Coulter Counter on. Turn the MCV Computer on by pressing the
power switch located on the left front of the instrument.
11.51 Threshold Adjustment
1. Set the controls on the Coulter Counter as follows:
Position
Control ZB or ZBI ZF
Upper Threshold Off
Lower Threshold 9 20
Separate/Locked Separate
Amplification \ 2
Aperture Current 1 1
Matching 20k
Gain Trim Mid range*
2. Place a sample of the Organic Calibration Material dilu-
tion on the sample platform of the Coulter Counter, im-
mersing the aperture tube and external electrode. Open
122
-------
the stopcock until pulses appear on the oscilloscope; then
i
close the stopcock. Record the count when the counting
stops. Repeat until 5 counts have been performed. Sum
the counts and divide by 5 to obtain the average count.
3. Turn the Lower Threshold control to about 22 and perform
another count. Perform several more counts and refine the
lower threshold setting until the count obtained is 50% ±
2000 of the average calculated in Step (2). Record the
Lower Threshold control setting.
4. Calculate the Threshold Factor. To do this, divide the
MCV of the Organic Calibration Material (60(jm3) by the
lower Threshold dial setting obtained in Step (3).
Example: Lower Threshold setting is 22.
Threshold Factor = ^> ?? ^ . .— = 2.73 umVdial
22 cell divisions ,. . .
division
5. Calculate and set the Lower Threshold to 25 \mz. To
calculate, divide 25 |jm3 by the Threshold Factor.
Example: ^-^ S2,^.'jm ,. . .— = 9 dial divisions**
H 2.73 pnr/dial division
6. Set the Threshold control on the rear of the Computer to
the same position as the Lower Threshold control on the
Coulter Counter.
7. Perform the operational check and Computer calibration,
Section 2-2.
123
-------
11.52 Operation Check and Computer Adjustment
1. Set the TEST/NORM switch found at the rear of the Computer
to NORM.
2. Perform a count of the Organic Calibration Material/Isoton
II or 1% NaCl dilution. Read the MCV display with the
MCV/RBC switch UP. Read the corrected count with the
MCV/RBC switch DOWN. Read the raw count from the Coulter
Counter. Look up the raw count on the coincidence cor-
rection chart for 100 urn aperature with 500 urn manometer
and read the corrected count. If these counts agree
within ±1.5% and the MCV is 60 ± 1.5% the system is ready
to analyze samples (see section 2-3). Otherwise, perform
Steps (3) through (7) below.
3. Set the TEST/NORM switch to TEST.
4. Set the MCV/RBC switch DOWN. Adjust the RBC calibration
control until the corrected count read from the coinci-
dence correction chart is displayed.
5. Set the MCV/RBC switch UP. Adjust the MCV calibration
control until 60 is displayed.
6. If the Hematocrit readout is used multiply the corrected
count times the MCV and adjust the Hct calibration control
until this value is displayed.
7. Repeat Steps (1) and (2).
124
-------
11. 53 Sample Analysis
Due to the differences in size distribution between the algae samples and
the calibration material the lower threshold setting must be decreased to
accomodate the smaller algae cells. The lower threshold should be set to
about 10.0 pm3 to eliminate debris and still count all of the cell population.
1. Calculate and set the Lower Threshold to 10.0 pm3. To
calculate divide 10.0 pm3 by the Threshold Factor.
Example:
10.0 pm3 . ,. , ,. . . **
2.73 pmVdial division = 4' dial ^visions**
2. Set the Threshold control on the rear of the Computer to
the same position as the Lower Threshold control on the
Coulter Counter.
* If the MCV unit can not be calibrated to read 60 pm3 at a gain setting of
5.0 (midrange) lower the gain to 3.0, obtain new reading and adjust to 60
pm3.
** Rounded to nearest whole number.
125
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing!
REPORT NO.
EPA-600/9-78-018
2.
3. RECIPIENT'S ACCESSION
TITLE AND SUBTITLE
The Selenastrum capricornutum Printz Algal Assay Bottle
Test: Experimental Design, Application, and Data
Interpretation Protocol
5. REPORT DATE
July 1977
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
William E. Miller, Joseph C. Greene and
Tamotsu Shiroyama
8. PER
PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory-Corvallis
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis. Oregon 97330
608a and/NE623
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
same
13. TYPE OF REPORT AND PERIOD COVERED
Finai
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
This report supercedes the Algal Assay Procedure: Bottle test (USEPA 1971).
16.ABSTRACT yhis document is the product of intensive research to improve and expand the
understanding of results obtained from the Algal Assay Procedure: Bottle Test (USEPA
1971) to enable investigators to define the stimulatory and/or inhibitory interaction(
of municipal, industrial and agri cultural wastes upon algal productivity in natural
waters.
This research was designed to determine:
The impact of nutrients and/or changes in their loading upon algal productivi
1.
2.
3.
4.
5.
Whether the growth response of Selenastrum capricornutum reflects the respons
of indigenous species;
The feasibility of the assay test protocol to evaluate heavy metals;
The capability of the assay to define the effect(s) of complex wastes; and
If the assay information can be applied to define and assist in the manage-
ment of real-world situation.
As a result of these research efforts the Selenastrum capricornutum Printz Algal Assay
Bottle Test: Experimental Design, Application and Data Interpretation Guide is offere
now for wider application in both eutrophication and toxicity problem aroas.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Selenastrum capricornutum Nutrient
limitation, heavy metal toxicity, complex
wastes, Algal growth potential, toxicity,
Eutrophication
b.IDENTIFIERS/OPEN ENDEDTERMS
COSATI Field/Group
18. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS (ThisReport)
unclassified
21. NO. OF PAGES
132
20. SECURITY CLASS (Thispage)
22. PRICE
I
unclassified
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
126
ft U.S. GOVERNMENT PRINTING OFFICE: 1978—797-624/222 REGION 10
------- |