Marine
Algal Assay Procedure
Bottle Test
Eutrophication and Lake Restoration Branch
National Environmental Research Center-Corvallis
DECEMBER 1974
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Specific mention of trade names or commercial products
does not imply or constitute endorsement or recommenda-
tion for use by the U. S. Environmental Protection Agency.
Cover photo of Slletz Bay, Oregon, courtesy of Dr. larry S. Slotta, Ocean
Engineering Program, Oregon State University. Corvallls. Oregon 9733J.
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MARINE ALGAL ASSAY PROCEDURE: BOTTLE TEST
Eutrophication and Lake Restoration Branch
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon 97330
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
December 1974
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TABLE OF CONTENTS
FOREWORD i
INTRODUCTION 1
PRINCIPLE 2
PLANNING AND EVALUATION OF ASSAYS, SELECTION OF
TEST WATERS, DETERMINATION OF LIMITING NUTRIENTS .... 3
APPARATUS 4
Sampling and Sample Preparation 4
Culture and Incubation 4
General 6
Optional 6
SAMPLE COLLECTION, TRANSPORT, PREPARATION AND STORAGE . . 7
Collection 7
Transport 7
Preparation 7
Storage 8
SYNTHETIC ALGAL NUTRIENT MEDIUM 8
Basal Medium 8
Stock Solutions 10
Reference Medium 10
INOCULUM 10
Test Algae 10
Source of Test Algae 11
Maintenance of Stock Cultures 11
Preparation of Inoculum 11
Strength of Inoculum 12
TEST CONDITIONS 12
Temperature 12
Illumination 12
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TABLE OF CONTENTS (Cont'd)
Page
PROCEDURE 12
Preparation of Glassware 12
pH Control 13
Growth Parameters 13
SPIKES 13
Auxiliary Spikes 15
DATA ANALYSIS AND INTERPRETATION 15
APPENDICES 17
Autoclaving 18
Flask Closures 18
Taxonomy 20
Inoculum Preparation 20
Data Reduction Equation 21
Inoculum Strength 24
Laboratory and Field Sample Data 25
Identification of Growth Limiting Nutrient 26
References 39
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LIST OF ILLUSTRATIONS
Figure Page
1 Inocul urn Strength Curves 27
2 Dunallella Phosphorus/Salinity Plot 28
3 Dunal lei la Mi trate-N/Sal i ni t.y Plot 29
4 Dunallella Anmonia-N/Salinlty Plot 30
5 Dunal iella Phosphorus Growth Response 31
6 Dunallella Nitrate-N Growth Response 32
7 Dunaliella Ammonia-N Growth Response 33
8 Dunaliella Southern Oregon Estuary Sampling Sites. 35
9 Southern Oregon Estuaries, Growth Limiting
nutrients 36
10 Southern Oregon Estuaries, Growth Limiting
Nutrients 37
LIST OF TABLES
1 Dunallella biomass per unit of nutrient 34
2 Yaquina Bay, Oregon, Bioassay Results 38
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FOREWORD
The success and widespread acceptance of the freshwater Algal
Assay Procedure: Bottle Test, has prompted the Eutrophication and
Lake Restoration Branch to develop a companion procedure of the test
to deal with problems of assessment of cultural eutrophication in
estuarine and marine coastal situations.
The developmental work at the Pacific Northwest Environmental
Research Laboratory, Corvallis, Oregon, consisted of choosing suitable
algal species, investigations to provide necessary background physiological
data, and field testing the process. It was necessary to find algal
species that had wide salinity tolerance, low or predictable nutrient
carry over, simple evaluation characteristics, good replicability,
and predictable growth response to various nutrient concentrations.
This work has been under the immediate direction of William E.
Miller and David T. Specht. To them goes much credit for effectively
developing the procedure.
Thomas E. Maloney, Chief
Eutrophication and Lake Restoration Branch
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MARINE ALGAL ASSAY PROCEDURE: BOTTLE TEST
1. Introduction
In February 1969, the Joint Industry/Government Task Force on
Eutrophication published the Provisional Algal Assay Procedure (PAAP).
Shortly after publication of the PAAP, a group consisting of government,
university and industrial laboratories undertook a comprehensive research
program to improve and evaluate it. While the PAAP consisted of three
fundamental test procedures, the first phase consisted of refining the
Bottle Test. After more than two years of intensive research and
development, the Algal Assay Procedure: Bottle Test was published in
August 1971.
During the course of evaluating the Bottle Test, an inter-
laboratory precision test was carried out by eight laboratories. The
results of this was published in October 1971 and entitled "Inter-
Laboratory Precision Test."
The Algal Assay Procedure Bottle Test has received widespread
acceptance and it was readily recognized that a similar test should
be developed to study eutrophication problems in estuarine and marine
coastal waters. Although the laboratory procedure is ready for routine
use, as was the case with fresh-water test, further evaluation relating
to specific field situations will be necessary (Specht and Miller, In
Press; Specht, In Press).
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2. Principle
The principle of this marine version of the Algal Assay Procedure
is essentially the same as previously published, but will be outlined
here.
The assay is based on LiebiVs Law of the Minimum, modified by
rate considerations [see O'Brien (1972); Holmes (1973); Kelley and
Hornberger (1973); O'Brien (1973)] in which the biomass produced in a
given amount of time (rate) is related to the available concentration
of the limiting nutrient.
The assay has been designed to assess receiving waters of
varying salinity as to nutrient status, biostirnulation potential,
and sensitivity to change in nutrient concentration.
The test is intended to identify algal growth-limiting nutrients,
biologically determine their availability, and quantify the biological
response to changes in concentration. It is anticipated that development
work in the future will show the various inhibitory effects of certain
pollutants on algal growth potential.
These measurements are made in a uniform manner by inoculating a
batch of test water with a selected test alga and determining algal
growth according to an established test protocol.
2.1 The maximum specific growth rate is related to the concentration
of the rate limiting nutrient present. The maximum standing crop is
proportional to the initial amount of the limiting nutrient available.
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2.2 All comparative growth responses should be statistically
analyzed and significant levels of differences reported. For most
purposes a 95 percent significance level can be considered statis-
tically significant.
3. Planning and Evaluation
The specific experimental design of each algal assay must be
dictated by the actual situation. It is extremely important that
all pertinent ecological factors be considered in planning a given
assay to insure that valid results and conclusions are obtained.
As a minimum, the following specifics must be considered by each
investigator who plans to conduct algal assays for the purposes
listed above.
3.1 Selection of test waters - Water quality of estuaries
will vary greatly with time and location. Sampling programs should
be established so that meaningful data will be obtained.
3.11 Spatial variations - The influence of point sources
of nutrients or pollutant input on algal biomass production or growth
rate may be determined by sampling upstream and downstream from the
point source, taking tidal fluctuations into account.
3.12 Temporal variations - Water quality in an estuary
will vary not only with season, but with each tide. Sampling
schedules should be arranged to take into account this variation,
sanpling preferably at high water, or at both high water and the
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following low water. An effort should be made to determine whether
the estuary is stratified or well mixed and sampling schemes should
be modified to account for this potential variable.
3.2 Determination of limiting nutrients - Any of the essential
nutrients may be limiting to algal growth. Bioassays are limited to
examination of only a few nutrients which are likely to be limiting.
See Appendix 3 (in AAP, August 1971) for an example of an experimental
plan.
4. Apparatus
4.1 Sampling and Sample Preparation
4.11 Water Sampler - Non-metallic
4.12 Sample Containers - Sterilizable (borosilicate glass,
linear polyethylene, polycarbonate, or polypropylene, for autoclaving);
polyethylene "cubitainers" Sterilizable by acid washing.
4.13 Membrane filter apparatus - 123 mm filter transfer
pump, for use with .45ym porosity prewashed filters; or 47 mm standard
apparatus.
4.14 Autoclaving - Considered at this time to be too drastic
a treatment because of precipitation of salts.
4.2 Culture and Incubation
4.21 Culture vessels. Erlenmeyer flasks of good quality
R R
borosilicate glass such as Pyrex or Kimax . The same brand of glass
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should be used within a given laboratory. When trace nutrients are
D
being studied, special materials, such as Vycor , polycarbonate, or
coated glassware should be used.
For gas exchange considerations, a contact surface to volume
ratio should be used as follows:
40 ml in 125 ml flask
60 ml in 250 ml flask
100 ml in 500 ml flask
4.22 Culture closures - Culture flask closures are preferably
polyurethane foam plugs (Gaymar ), or can be loose fitting aluminum
foil or small inverted beakers. These must be tested for toxic effects
on algal growth.
4.23 A constant temperature room, or equivalent incubator
is needed to provide temperature control at 18° +_ 2°C.
4.24 Illumination - "cool white" fluorescent lamps to give
at least 250 ft-c (2152 lux), preferably 400 ft-c (4304 lux). Adjust
lighting to give +_ 10 percent illumination over the entire shaker
platform. Measure adjacent to liquid level in flask.
Note: The energy level output of a bank of six 48-inch "cool
white" fluorescent lamps (GE 40 watt, @ 60 Hz) was approximately 1300
p
uw/cm (range, 380-760 nm) at a distance of 26 3/4 inches, as measured
with an ISCO Model SRC spectroradiometer. Using the same measurement
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geometry, a Weston Model 756 Illumination Meter read 400 ft-c. All
reflecting surfaces were matte white.
Therefore, utilizing a calibrated illumination meter with a
foot-candle readout, one may, by adjusting the height of the lights,
2
achieve a known energy level output of 1300 yw/cm .
For further discussion of the problems of the differences in
absorption of light by photosynthesizing organisms and by man's eye
and their measurement, see Tyler (1973).
4.25 Light meter - flust be calibrated against a standard
light source or meter.
4.3 General Apparatus
4.31 Analytical balance, capable of weighing 100 gm with
a precision of ^ 0.1 mg.
4.32 Microscope - Good quality general purpose microscope
with illuminator.
4.33 Haemocytometer or plankton counting slide.
4.34 pH meter - Scale of 0-14 units with accuracy of +_ 0.1
pH unit.
4.35 Oven, dry heat, capable of temperatures to 120°C.
4.36 Centrifuge - Capable of centrifugal force of at least
1000 x g.
4.37 Spectrophotometer or colorimeter - For use at 600-750 nm.
4.4 Optional, but desirable equipment.
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4.41 Electronic cell counter.
4.42 Fluorometer.
4.43 Shaker table, capable of 110 oscillations per minute.
5. Sample Collection, Transport, Preparation and Storage
5.1 Collection - Use non-metallic water sampler and sterilizable
containers (see Section 4.11, 4.12). Containers should be pre-rinsed
with a portion of the sample to acclimate the interior surface to the
nutrient concentrations of the sample. Containers suspected of toxic
or nutrient contamination should not be used.
5.2 Transport condition - Fill containers to leave minimum air
space, refrigerate (ice) and keep in dark during transporation.
5.3 Preparation - Mix sample thoroughly and remove enough sample
for pre-filtration chemical analysis (if desired), and filter to remove
indigenous algae. Autoclaving is not recommended for marine or estuarine
samples at this time.
5.31 Membrane filtration - Removes indigenous algae and
detritus. Bioassay then determines growth limiting nutrient not taken
up by indigenous organisms removed by filtration. Pretreat 0.45 pro
membrane filter by passing at least 50 ml double glass distilled water
through it (47 mm filter) or one liter of water (123 mm filter).
Discard this filtrate. Proceed to filter the quantity of sample
needed under reduced pressure of 0.5 atmosphere or under pressure of
less than 1.5 atmosphere.
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If a great deal of suspended matter is present, a prewashed
prefilter may be used, or precentrifugation utilized.
5.4 Storage - Samples should be stored at 0-4°C in the dark,
excluding air bubbles in the container. If prolonged storage is
anticipated, samples should be prepared (filtered) first.
6. Synthetic Algal Nutrient Medium
6.1 Basal Medium: Modified Burkholder's* Artificial Seawater
(ASW) with NAAM levels of the following nutrients: N, P, Fe, and
Na2EDTA. Use Analytical Reagent or Reagent Grade chemicals.
Compound grams/I grams/4 1
NaCl 23.48 93.92
Na2S04 3.92 15.68
NaHC03 0.19 0.76
KC1 0.66 2.64
KBr 0.10 0.38
H3B03 0.03 0.10
MgCl2 • 6H20 10.61 42.44
SrCl2 • 6H20 0.04 0.16
CaCl2 • 2H20 1.47 5.88
H20 to 1,000 ml 4,000 ml
filter through prewashed 0.45 ym membrane filter.
*Ref. Burkholder, P. 1963. Some nutritional relationships among
microbes of the sea sediments and waters. In: Symposium on Marine
Microbiology, Ed. C. H. Oppenheimer. pp. 1133-1150. Thomas,
Springfield, Illinois.
8
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FOR DILUTION TO VARIOUS SALINITIES: (4 liter batches)
Salinity °/0o
35
30
24
20
16
12
8
5
ASW Stock, 1
4.000
3.43
2.74
2.29
1.83
1.37
0.91
0.57
H20 (glass distilled), 1
0.000
0.57
1.26
1.71
2.17
2.63
3.09
3.43
For any given final salinity, mix well, adding the following NAAM
levels of nutrients:
NaNO 102 mg/4-1 batch (4.2 mg N/l)
K2HP04 4.18 mg/4-1 batch (0.186 mg P/l)
Na2EDTA 1200 yg/4-1 batch (300 yg/1)
*NAAM trace metal mix (minus Fed J
Filter through 0.45 ym membrane filter, add AFTER filtration, sterilized
FeCl3, 384 ug/4-1 batch (33.05 ug Fe/1).
*Add the following: 0.0928 g H,BO,; 0.208 g MnCl? 4H 0; 0.016 g
O O c. L-
ZnCU; 0.714 mg CoCl2- 6H20; 0.0107 mg CuCl2 • 2H20; 3.63 mg Na
2H?0; make up to 500 ml., adding 1 ml of this concentrate to each
1iter of media.
Adjust to pH of 8.0 jf 0.1, if necessary.
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6.2 Stock solutions - Stock solutions of some salts may be made
up 1000 times the final concentration. Practically speaking, these
are N, P, Fe, EDTA, trace metals, Sr, Br. The remaining salts are
required in amounts that are impractical to hold in stock solutions.
6.3 Reference medium - Make up ASW to 20 °/00» store in dark at
4°C excluding air from the container. This concentration should be
used to raise the inoculum. The reference medium should be diluted
from 35 °/00 stock, according to the dilution schedule, to match the
salinity of the sample.
7. Inoculum
7.1 Test Algae
7.11 Dunaliella tertiolecta Butcher (DUN clone).
7.12 Thalassiosira p_seudonana Hasle and Heimdal,* (CN clone)
(Cyclotella nana Hustedt)
*This organism is currently being evaluated for use. If used,
add Si and vitamins to stock medium:
0.6 mg Na2Si03- 9H20/1
1.0 ml vitamin mix/1
7.121 Preparation of vitamin mix: Stock - 0.1 rug/ml
Biotin (dissolve by warming, if necessary). 0.1 mg/ml B,,>. Make 10
ml in double glass distilled water, keep frozen and sterile.
10
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Take 1 ml vitamin mix, add 20 mg thiarnine hydro chloride to
100 ml double glass distilled water. Dispense into 1 ml ampoules (or
scalable containers). Autoclave 5 minutes, store frozen. Use 1 ml/1
final solution. (From Curl, H. W. 1971). Preparation of Basic Culture
Media. Mimeograph copy. Oregon State University Dept. Oceanography.
7.2 Source of test algae - Available from the Eutrophication
and Lake Restoration Branch, Pacific Northwest Environmental Research
Laboratory, NERC-Corvallis, EPA, 200 SW 35th Street, Con/all is, OR 97330.
7.3 Maintenance of stock cultures.
7.31 Media. (See Section 7 and 8.12)
7.32 Incubation conditions. (See Section 4.24) 18° ^2°C
under continuous illumination of 400 ft-c (4304 lux +_ 10 percent for
Dunaliella).
10° ± 2°C under continuous illumination 550-600 ft-c (5900-6500
lux) ± 10 percent for Thai assipsvra.
7.33 Transfer of cultures - Transfer under aseptic conditions
at least every seven days from previously unopened stock flask, 1 ml
into 100 ml new media.
7.34 Age of Inoculum: Use cultures 5-7 days of age,
preferably on the younger side so cells are in or near log phase of
growth.
7.4 Preparation of Inoculum - Cells from stock culture should be
centrifuged in sterile centrifuge tubes and the supernatant discarded.
11
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The sedimented cells should be resuspended in filter sterilized 20 °/00
ASM less N, P, EDTA, trace metals and Fe, and again centrifuged.
Discard the supernatant and again resuspend the pellet in the 20 °/00
ASM as before, diluting to the appropriate concentration for the
inoculum, (see Appendix 7.5 for example with typical calculations.)
7.5 Strength of inoculum - The prepared cell suspension (Appendix
7.5) should be counted and adjusted by dilution to 10,000 cells/ml so
that a 1 ml inoculum iinto 99 ml of sample + spikes will give a final
concentration of 100 cells per ml. (Approximately 0.02 - 0.03 mg/1
dry weight, see Appendix 7.5).
8. Test Conditions
8.1 Temperature - 18° - 20°C
8.2 Illumination - Continuous "cool white" fluorescent lighting
400 ft-c (4304 lux) +_ 10 percent for Dunaliella; 550-600 ft-c (5900-6500
lux) +_ 10 percent for Thalassiosira (see Section 4.24).
8.3 Shakers - To facilitate gas exchange, Puna!Jell a should
either be continuously shaken at 110 oscillations/minute or kept static
and the flasks swirled by hand twice/day (continuous shaking is
desirable for uniformity). Thalassiosira should not be shaken, but
kept static and swirled once/day by hand (i.e., as when mixing prior
to sampling).
9. Procedure
9.1 Preparation of Glassware - All glassware associated with
12
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the test should be washed with phosphate-free detergent or sodium
carbonate and rinsed thoroughly with tap water. This is followed by
a rinse with 10 percent by volume of reagent grade hydrochloric acid
(HC1); glassware is filled momentarily with the HC1 solution, swirled,
dumped into the next flask, filled with a 10 percent by weight reagent
grade sodium carbonate (Na2C03) to neutralize the glass surface, then
rinsed 5 times with deionized water, oven dried, inverted on racks at
j^
105°C, capped with aluminum foil or stoppered with foam plugs (Gaymar )
and autoclaved at 15 psi for 15 minutes.
Before dispensing, flasks should be prerinsed with an aliquot of
the medium or test water to be used, to "acclimate" the glass surfaces
to the concentrations of metals and nutrients in these waters.
9.2 pH Control - Sea water is usually a good buffer, and pH
problems should not occur except perhaps with the very low salinity
estuarine waters (below 5 °/00). Control of pH is accomplished by
using optimal surface to volume ratios and adequate mixing to allow
availability of carbon dioxide.
9.3. Growth Parameters - Two parameters are used to describe
the growth of a test alga in the Bottle Test: maximum specific growth
rate and maximum standing crop. Either or both of these parameters
may be determined, depending on the objectives of the particular assay.
10. Spikes
The quantity of cells that may be produced in a given medium is
13
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limited by the substance that is present in the lowest relative quantity
with respect to the needs of the organism. If a quantity of the
limiting substance were added to the medium, cell production would
increase until this additional supply was either depleted or until
some other substance became limiting to the organism (see Appendix 10).
Additions of substances other than that which is limiting would yield
no increase in cell production. Nutrient additions may be made singly
or in combination, including wastewaters, and the growth response may
be compared to unspiked controls to identify those substances which
limit growth rate or cell production. The selection of spikes, e.g.,
nitrogen, phosphorus, iron, sewage effluents, etc., will depend on the
answer being sought.
In all instances, the volume of the spike should be as small as
possible. The concentration of spikes will vary and must be matched
to the waters being tested. Two considerations should be taken into
account when selecting the concentration of spikes: (1) the
concentration should be kept small to minimize alterations of the
sample, but at the same time it should be sufficiently large to yield
a potentially measurable response; and (2) the concentration of spikes
should be related to the fertility of the sample. To assess the effect
of nutrient additions, they must be compared to an unspiked control of
the test water. If the control is quite fertile, cell production will
be high and flask-to-flask variations in the controls might mask the
effect of small additions of the limiting nutrient (see Appendix 10).
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10.1 Auxiliary Spikes - In addition to spikes for the purpose
of determining stimulatory or inhibitory effects on algal growth in
test waters, it is sometimes necessary to check for the possibility
that the test water contains some toxic material which could influence
results. To check for toxic materials, the test water may be spiked
with the elements in complete synthetic medium (see Section 6). If
no increase in growth occurs, the presence of toxic materials is
suspected. In some situations, dilution of the sample or the addition
of a chelator will eliminate toxic effects.
11. Data Analysis and Interpretation (See Algal Assay Procedure:
Bottle Test, August, 1971, pp. 21-23)
11.1 Introduction - The fundamental measure used in the
Algal Assay: Bottle Test to describe algal growth is the amount of
suspended solids (dry weight) produced and this is determined
gravimetrically. Other biomass indicators such as those shown in 9.6
may be used; however, all results presented must include experimentally
determined conversion factors between the indicator used and the dry
weight of suspended solids.
Several different biomass indicators should be used whenever
possible because biomass indicators may respond differently to any
given nutrient limiting condition.
11.2 Reference Curves - Results of spiking assays should be
presented together with the results from two types of reference
samples; the assay reference medium and unspiked samples of the
15
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water under consideration. Preferably the entire growth curves
should be presented for each of the two types of reference samples.
The results of individual assays should be presented in the form of
the maximum specific growth rate (with time of occurrence) and
maximum standing crop (with time at which it was reached), (see
AAP, August 1971).
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APPENDICES*
*Each Appendix is numbered to correspond with that Section to which
its contents are related. Some Sections do not have a corresponding
Appendix.
17
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APPENDIX 4
4.14 Autoclaving - Several alternatives to autoclaving are being
investigated to avoid the irreversible precipitation problems caused by
heat input. The most promising method is that of explosive decompression.
This is accomplished by loading the sample into a large (1 gallon
capacity) chemical "bomb," charging with dry nitrogen (Np) at pressures
2
not to exceed 2,000 Ibs/in , and allowing the contents to come to
equilibrium. The contents of the bomb are released under controlled
procedures into a container. Under ambient pressure, the cellular
material contained therein releases the absorbed nitrogen by rupturing
cell walls, effectively sterilizing the sample with respect to algae.
Some bacterial and yeast spores are capable of withstanding such
treatment, but no algae have been reported as surviving. No available
nitrogen is contributed to the sample, there is no heat input (in fact,
upon release, the sample undergoes adiabatic expansion), and the
discharged sample may be kept under a blanket of inert nitrogen.
4.22 Flask Closures - Closures must demonstrate the following
properties:
maintain integrity of culture
survive autoclaving
provide uniform gas exchange
be non-toxic to the test species
18
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While several types of closures have proven to be toxic,
primarily by volatiles released during autoclaving, some specific
types have proven satisfactory:
aluminum foil
D
Delong closures (stainless steel)
p
Gaymar polyurethane foam plugs
The metal closures have the disadvantage of blocking or reflecting
light. Other types or brands of plastic foam plugs must be tested for
toxicity.
19
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APPENDIX 7
7.11 Taxonomy:
Dunaliella tertiolecta Butcher (DUN clone) is a green uni-
cellular flagellate, Class Chlorophyceae, Order Volvocales, Family
Polyblepharidaceae. Cells are ovoid, 5-8 x 10-12 ym, with two long
flagella at the anterior end and have one cup shaped chloroplast,
with a single pyrenoid, at the posterior end.
Asexual reproduction takes place by longitudinal division,
although the alga may occasionally reproduce sexually, by isogamy,
producing up to 16 zoospores. Under growth stimulating conditions,
asexual reproduction prevails.
Thalassiosira pseudonana Hasle and Heimdal (CN clone)
Cyclotella nana Hustedt is a centric diatom, Class Bacillariophyceae,
Order Centrales, Family Discoideae, 4-9 ym in diameter. The usual
reproductive process is asexual fission, but sexual fusion by
anisogamy may occur. Although succussive vegetative division may
tend to reduce cell size, periodic auxospore formation will restore
cell size.
7.4 Preparation of Inoculum - Cells are obtained from a stock
culture, preferably in log phase of growth. Take, for instance, 40 ml
of stock culture, pour into sterile centrifuge tube, cap, and centrifuge
for 10 minutes at 2,000 rpm. Decant the supernatant, refill to mark
with filter (0.45 ym) sterilized 20 °/00 ASW less N, P, Fe, EDTA, and
20
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micronutrients (make up and keep in separate container for this
purpose), and centrifuge again for 10 minutes at 2,000 rpm. Again
°/
decant the supernatant, and refill with 20 ' °° ASW diluent. Take a
1 ml subsample of this preparation and count.
For example: Counting with an electronic particle counter, at a
dilution of 1 ml sample to 99 ml of 1% NaCl electroyte (1:100 dilution),
the machine counts the number of cells in 0.5 ml of the 1:100 dilution:
3
Count MCV (ym ) Machine settings: Amplification
Aperture: ~\/h
6217 81
6182 78 Background count in filtered saline
electrolyte - 10 -
6068 78
6074 77
6039 76
390/5 = 78+1.9
Enter these data into the following data reduction equation:
([a-x] mcvg) + ([b-x] mcvb) + ... ([n-x] mcvn)
~" ' n
•(l+[2.52 x 10"6{([a-x] mcva) + ([b-x] mcvb) + ... ([n-x] mcvn)}])
n
• Y- (dry weight factor) = calculated dry weight
where: a, b,...n = numerical cell counts
mcv , mcv, ,...mcv = associated mean cell volumes
a D ^n
in ym
(l+[2.52 x 10" ]) = coincidence correction factor
Y = dilution factor (multiplier)
X = background count
-7 3
(dry weight factor) = 8.66 x 10 mg/ym cell volume,
for Dunaliella tertiolectaApproximately
the same for Thalassiosi ra pseudonana.
21
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(The second part of the expression is a coincidence correction
factor to account for the statistical probability of more than one cell
passing through the orifice at a given time and appearing electronically
as one cell twice as large as it really is. 2.52 x 10" is the factor
for a lOOym orifice; other orifice sizes require different numbers,
which can be supplied by the manufacturer of the counter.)
This expression, which may be set up on a small programmable desk
calculator, can be arranged to yield the corrected cell count and the
calculated dry weight.
Corrected count for this example: 1,240,000 cells/ml in stock
culture or 83.8 mg/1 calculated dry weight.
Needed: 100 cells/ml; 100 mis/flask; and, for example, 100 flasks
or 10,000 cells/flask x 100 flasks to total 1,000,000 cells in 100 ml
of inoculum.
Since there are 1,240,000 cells/ml in the stock preparation:
1'240'QQO = °'806 ml of stock PreParation
Add 0.81 ml of stock to a 100 ml volumetric flask, and top up
with 20 °/oo ASM diluent. After thorough shaking to mix, withdraw a
1 ml sample and count.
Since this is a more dilute cell suspension, dilute only 1 + 9
(1:10).
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Count MCV (y3) Background - 10 -
655 82
642 82
585 72
620 71
659 70
75 + 6.1
= 12,500 cells/ml
This amounts to a final flask concentration of 125 cells/ml,
which is about 20 °/0 too high. To correct, calculate:
12,500 x 99 (mis inoculum left in flask)
1,238,000 (Total number of cells left in flask)
1,000,000 (Number of cells desired)
214,000/12,300 (Excess cells/ml concentration)
= 19.00 ml (amount to remove from inoculum flask)
Remove 19.00 ml from the inoculum flask and top up with the
20 °/00 ASW diluent, withdraw another 1 ml sample, dilute 1:10
and count:
MCV (ym3)
75
73
76
69
70
= 10,189 cells/ml 70 + 3.0
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-------
The final flask concentration at inoculation is now 102 cells/ml
or, 0.064 mg dry weight/liter.
The proper concentration may be obtained after the first dilution
and no other manipulations are necessary. This, however, is usually
not the case.
7.5 Strength of inoculum - Because nutrient carryover in an
inoculum could prejudice results of the bioassay in more austere
water samples, the initial inoculum level was reduced from 1,000
cells/ml (0.3 mg/1 dry weight) to 100 cells/ml (0.03 mg/1 dry weight).
Although this introduces a detectable lag in growth response, growth
rate appeared unaffected and the final yield was identical at day 10
or 12 (see Figure 1). The inoculum was dispensed in a 1 ml volume.
24
-------
APPENDIX 10
10.1 Laboratory and Field Sample Data
The biomass response of the test alga (Dunaliella) at day
10 is graphically illustrated in Figures 2, 3, and 4 in the form of a
3-dimensional response-surface. These responses indicate that, in ASW,
Dunaliella will show significant growth response at 2.5 to 50 yg/1
phosphorus (Figure 5), 10.0 to 1,000 yg/1 nitrate nitrogen (Figure 6),
and 10.0 to 1,000 yg/1 ammonia nitrogen (Figure 7), in 5 °/00 to 35 °/0<
salinity.
In ASW, 5 °/oo to 35 °/00 salinity, Dunaliella produces an
average of 1.08 mg dry weight per yg of phosphorus, 0.03 mg dry
weight per yg of nitrate nitrogen and 0.08 mg dry weight per yg
of ammonia nitrogen (see Table 1).
Dunaliella is consistent in its response, showing excellent
replication. All experimental runs were performed in triplicate.
An example of this consistency can be illustrated in the result of
an investigation of nitrilo-tri-acetic acid, (NTA), as a potential
nitrogen source in natural water samples. In addition to control,
four levels of NTA, 0.05 to 1.0 mg/1 (expressed as N), were added to
water samples from six Oregon estuaries (five of which were nitrogen
limited at the time) in triplicate. NTA neither stimulated nor
inhibited growth in any sample in the time period; a t-test (13
degrees of freedom) showed no significant difference between an
increase in NTA-N and dry weight produced. The normalized standard
deviation for the entire run was less than ;+ 15 percent.
25
-------
APPENDIX 11
11.4 Identification of growth-limiting nutrient.
A set of samples taken July 25, 1973, from five southern Oregon
Coastal estuaries (Figure 8) at high water and assayed with
Dunaliella showed that all except the Umpqua River were growth
limited by nitrogen. The Umpqua River station (11.6 °/0o salinity)
responded significantly to phosphorus spikes, but not to nitrogen
(see Figures 9 and 10) (Specht and Miller* In Press).
In nitrogen-limiting situations, the growth rate of the test
algae is significantly greater when ammonia nitrogen is added than
when nitrate nitrogen is added. However, if allowed to incubate for
a sufficient period of tine, there is no significant difference between
tiie final dry weigiit yields obtained with either of the nitrogen sources.
Assays performed on a set of samples taken on the low and following
high tides in the Yaquina Uay estuary, February 9, 1974, illustrates the
ability of the assay to define the boundary between nitrogen-limited
seaward water and phosphorus-linn'ted landward water as it moves in and
out of the bay. Table 2 (Specht, In Press) shows the control dry
weights, growth-limiting nutrient, salinity, phosphorus and nitrogen
levels, and the dry weight produced per unit of the limiting nutrient
as compared with the biomass produced in ASU (adjusted for salinity;
see appendix 10.1, paragraph 2). It is interesting to note the degree
of dependence and linearity of growth upon the limiting nutrient
concentration, rather than upon salinity.
26
-------
%o ASW + Full NAAM nutrients
inoculum strength cells/ml
o 100/ml
250/ml
1000/ml
12 16 20 24
10
0 4
Growth response of Dunaliella tertiolecta
20%« ASW+FutlNAAM nutrients
inoculum strength cells/ml
100/ml
250/ml
a 1000/ml
10
20 24
DAYS
Growth response of Dunoliella tertiolecta
16 %o ASWt Full NAAM nutrients
inoculum strength cells/ml
o lOO/ml
250/ml
a lOOO/ml
8 12 16 20 24
10
Growth response of Dunaliella tertiolecta
35%o ASW+ Full NAAM nutrients
inoculum strength cells/ml
o 100/ml
250ml
O 1000/ml
10
20 24
DAYS
Growth response of Dunaliella tertiolecta
27
-------
to
00
Growth of DunaliellQ at various salinities and
phosphorus concentrations in ASW.
Dry weight at day 10.
100.
r = .818
slope 1069.698
slope 352.816 r=.940
16°fr
1025,819
= .961
" fr
slope 1228043
r= .986
16°fr
r ••
n.6
r=.882
O.05
°0
Fig. 2
-------
Growth of Dunaliella at various salinities and
nitrate nitrogen concentrations in A5W.
Dry weight at day 10.
PO
u>
slope 8.479
r=.983
16°fr
slope 9.445
r=.978
slrpe 10.195
r=.981
I6°fr
slope 9.972
r--.977
16°tr
1,0
Fig. 3
-------
Growth of Dunaliella at various salinities and
ammonia nitrogen concentrations in ASW.
Dry weight at day 10.
w
o
1OO,
r = .606
lO°fr
slope 40 34
-------
I03 p~l—I—I—I—I—I—I—]—I—I—I—I—I—I—I—I—I—I—I—I—I—I—l~:
5%o ASW
+NAAM nutrients .
-phosphorus
O + O mg P/L
O -K0025mg P/L
n -t-.OOSmg P/L :
A + .Ol mg P/L
O -t- ,025mg P/L -
V -(-.OS mg P/L -
MA 022373
.1,1,1.
IOS L I I I | I I I | I I T | I I I
8
12
16
2O
24
DAYS
Growth response of DunaliellQ tertiolecta
I03 L I I I I
10*
£10°
O
10
20%«. ASW
+ NAAM nutrients
-phosphorus
+ 0 mg P/L
-K0025mg P/L
-KOOSmg P/L :
+ .01 mg P/L
f .025mg P/L
+.05 mg P/L -
10
-2
MA 022373
!2
16
2O
24
DAYS
Growth response of Dunaliello tertiolecta
16 %„ ASW
+ NAAM nutrients
- phosphorus
O +0 mg P/L
O t.0025mg P/L.
a + .OO5 mg P/L :
A + .01 mg P/L
O + .025mg P/L
V 4.05mg P/L
MA 022373
12
16
20
24
DAYS
Growth response of Dunaliella tertiolecta
IOJ
to1
10
.-2
_L
e
35%o ASW
+NAAM nutrients I
-phosphorus
O +0 mg P/L
O +.O025mg P/L
a -KOOSmg P/L :
A + .Ol mg P/L
O -t- .O25mg P/L -
V +.O5mg P/L -
MA 030973R
j—i—I—i—i—i I i i
12
16
20
24
DAYS
Growth response of DunaliellQ tertioiecta
Fig 5
3 I
-------
12
5%. ASW
+ NA AM nutrients'
- nitrogen
low NOj -N-
O t 0 mg N/L
O + J0lmg N/L .
n + O5mg N/L
A + .10 mg N/L -
Of.SOmg N/L
V + I.Omg N/L
MA 03O973
10s FT
I01
xlO'
IOC
10
16
2O
24
10"
16 %o ASW
+ NAAM nutrients ^
- nitrogen
low NOj -N-
Q + 0 mg N/L
G +.OImg N/L
D + .05 mg N/L
A+ .10 mg N/L
OH- .50mg N/L "
V + I.Omg N/L "
MA O3O973
, 1 , . , I ,
12
16
20 24
DAYS
Growth response of Dunoliella tertiolecta
DAYS
Growth response of Dunaliella tertiolecta
20%» ASW
+ NAAM nutrients
- nitrogen
low NO; -N-
O + O mg N/L
O +.OImg N/L
+.05mg N/L
A + . 10 mg N/L
O + .50 mg N/L
V + 1.0 mg N/L
MA 03O973
I . .1
10s ,.
Fig. 6
1C
35 %o ASW
+ NAAM nutrients
- nitrogen
low NO: -N-
O + O mg N/L
O *X>lmg N/L
+X)5mg N/L
A+ .|O mg N/L
O •*• .50 mg N/L
V + 1.0 mg N/L
MA 03O973
10
DAYS
Growth response of Dunoliello tertiolecta
2O 24
DAYS
Growth response of Dunoliella tertiolecta
32
-------
5%oASW
+NAAM nutrients .
-nitrogen
lowNH^-N-
O + O mg N/L
O * JOI mg N/L _
Q + .05mg N/L :
A -t-. 1 mg N/L _:
.5mg N/L -
V + I Omg N/L "
I03n-r-r
«02
vIO'
£10°
a
10
!6%oASW
+ NAAM nutrients -
- nitrogen
lowNHj-N-
O +0mg N/L
O + Q\ mg N/L _
Q + .05mg N/L :
A +. I mg N/L -
O + .5mg N/L
V + l.0mg N/L
MA 032373
, I , . , I ,
0 4
B
12
20
24
DAYS
Growth response of Puna lie! la tertiolecta
DAYS
Growth response of Dunoliella tertiolecta
10s FT
IOZ
I
H
*
10'
20%«ASW
•t-NAAM nutrients -
-nitrogen
lowNH^-N-
O + Omg N/L
O + .01 mg N/L
Q + .05mg N/L
A + . | mg N/L
O + .5mg N/L
V + I.Omg N/L
MA 032373
. I i i I ,
2O
24
DAYS
Growth response of Dunaliella tertiolecto
Fig. 7
33
I03
I02
vIO'
10°
10
JL
0 4
_L
S
J.
35%oASW
-t-NAAM nutrients"
-nitrogen
lowNH^-N-
O +Omg N/L
O + .01 mg N/L
Q + .O5mgN/L
A + . | mg N/L
O -t-.Smg N/L
V + I.Omg N/L
MA 032373
12
_L
_L
1C
20
24
DAYS
Growth response of Dunoliello tertiolecta
-------
TABLE 1
BIOMASS PRODUCED PER UNIT OF NUTRIENT BY DUNALIELLA
Nutrient
Salinity
5 %0
16%0
20%0
35%0
Average*
mg
P
0.557
+0.158
0.930
+0.240
1.170
+0.164
1.129
+0.232
1.076
AT DAY 14 IN
dry weight/yg of
N03- - N
0.0096
+0.0014
0.0308
+0.0394
0.0331
+0.0379
0.0315
+0.0361
0.0318
DEFINED MEDIA
nutrient P:N ratios
NH + - N P:NO - - N P:NH/ - N
4 3 *
0.0747 1:58 1:7.5
+0. 0075
0.0844 1:30.2 1:11.0
+0. 0058
0.0766 1:35.3 1:15.3
+0.005
0.0765 1:35.8 1:14.8
+0.0193
0.0796 1:33.8 1:13.5
*Average is calculated from the 16 %0, 20%0, and 35%0 data only.
34
-------
NORTH BEND
COOS BAY
-------
1O FT
I I 1 FT"
10'
I
10"
-
10
O
Coos Bay
at North Bend, Or.
O Control
O »CX>5mg RfL
O •lOmg NO4-N-/L
A .1 Omg NH? -N-/L
O »OO5mg RfL»1Omg. NO|-N-/L
2O 24
12 16
DAYS
Growth response of Dunaliella
tertiolecta
10'
PKD
1O
'
10
Coos Bay at
Horsefall Road Bridge _
O Control
O .OOSmg.RL
Q .1Omg
A «1.0mg
• OOSmg P/L »l.Omg. NH4 -N-/L
, I . . , I , , , I ,
:
2O 24
4 8 12 16
DAYS
Growth response of Dunaliella
tertiolecta
102
—T\ — .—» I
CD
110°
10
10
Siuslaw River
at Florence, Or
O Control
O •OOSmg RL
D «10mg NO4 -N-/L
A *1.0mg NH? -N-/L
O •OO5mgRfl_-10rrg
W »OO5nng Pn_-1Omg
i . , I , . i ,
2O 24
4 8 12 15
DAYS
Growth response of Djjnaliella
> i • . -^'
10
^^1
tn
@ 10°
-
10"
10
Umpqua River
at Reedsport,Or
O Control
O *OO5mg FKL
Q .l.Omg NOo -N-/L
A «1.0mg NIHt -N-/L
O »CX35mg F¥L«lOmg NC>3-N-/L
^ •QO5mgP/L«10mg NH4-
i 1 i i I ,
O 4 8 12 16 2O
DAYS
Growth response of Dunaliella
tertiolecta
:
I
Fig. 9
36
-------
Alsea River
at Waldport, On
O Control
O *Q05mg PIL
lOmg NO§ -N-/L
1.Omg NHl -N-/L
O.OSmg. Pit»10mg. NCft-N-/L
•O.O5mg.RL«1.0mg NH4-N-/L
, I , , I ,
o . : 10
DAYS
Growth r^ponse of 0
tertiolecta
.
1O"
10"
G
Yaquina River
at Newport, Or
O Control
O 'QOfnng.FyL
D *1.0mg. NO? -N-/L
£> «1.0mg. NH^ -N-/L
O *Q05mg.P/L-1.Omg.NO3-N-/L
V »O.O5mg.P/L»l.Omg.Nf-£-N-/L
, , i I , , , I , , , I ,
3
20 24
12
DAYS
Growth response of Dynaliella
tertiolecta
O Coos Bay at North Berxj
O Coos Bay at Horsetail Rd. Br ^
D Umpqua River at Reedsport
Siustaw River at Florence
<> Alsea River at Waldport
Yaauina River at Newport
Fig. I0
DAYS
Growth response of
37
Dunaliella
-------
TABLE 2
YAQUINA BAY, OREGON
Algal assay growth response and associated parameters
from low water-high water samples collected 02/9/'74
(Surface grab samples, membrane filtered)
00
Sampling Station
Treatment
Control dry wt.,
day 10, mg/lt
+ 0.05 mg P/l
+ 1 .0 mg N/l
Growth limiting
nutrient
Salinity, %a
Ortho-P
N (NO,+NO^NH.)
mg/1 * J *
mg dry wt/ug P
mg dry wt/pg N
mg dry wt/pg of
limiting nutrient
1n ASW (Specht &
Miller, in press)
adj. for salinity
Linear regression of
parameter vs dry wt.
P (of P limited samples)
N (of N limited samples)
Salinity
OSU Dock
low tide
28.9
37.3*
30.5
P
19.0
0.024
0.53
1.20
0.054
1.11
OSU Dock
high tide
16.8
17.1
38.8*
N
29.0
0.027
0.234
0.621
0.071
0.077
Slope
2090.0
42.1
0.359
Sally's Bend
low tide
21.8
46.2*
21.6
P
13.8
0.021
0.685
1.04
0.031
0.810
Intercept
-21.9
7.9
13.1
Sally's Bend
high tide
19.2
17.3
36.4*
N
28.2
0.027
0.263
0,710
0.072
0.076
Correlation
coefficient (r)
0.996*
0.990*
0.392
River Bend
low tide
7.35
43.8*
8.1
P
6.7
0.014
0.818
O.S24
0.008
0.614
t-test,
of (r)
35.5*
19.2*
1.71
River Bend
high tide
27.5
34.7*
31.3*
P (N)
22.8
0.024
0.478
1.15
0.057
1.16
sign. 1 degrees
of freedom
10
7
16
*1ndicates statistically significant difference
tall dry weights are geometric means of triplicate samples
-------
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