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

-------
          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

-------
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

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                              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

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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

-------
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

-------
                                   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

-------
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

-------
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

-------
          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

-------
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

-------
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

-------
     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

-------
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

-------
          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

-------
          (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

-------
     (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

-------
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.

Theis, T. L.,  and P. C. Singer.  1973.  The stabilization of ferrous iron by
     organic compounds in natural waters,  p. 303-320 lr\ P. C.  Singer [ed. ],
     Trace metals and metal-organic  interactions in natural waters.  Ann Arbor
     Sci.

Thomas, W. H. , and A. N. Dodson.  1968.  Effects of phosphate concentrations
     on cell division and yield of a tropical oceanic diatom.  Biol. Bull.
     134:199-208.

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-
     bial. 74(1):77-81.

Denison, J. R.  1974.  Limiting nutrient tests used in an  investigation of
     factors controlling phytoplankton development.  Water Treat.  Exam.
     23(l):52-75.

Dye, C., J. Hand, D. Jones, and L. Ross.  1978.  Determination of  critical
     nutrient levels in Florida lakes using algal assays.  Presented at 41st
     Annual Meeting ASLO, 19-22 June 1978, Victoria, British Columbia,  Canada.

Elfving, E., A.  Forsberg, and C. Forsberg.  1975.  Minitest method for  mon-
     itoring effluent quality.  J. Water. Pollut. Control. Fed. 47(4):720-726.

Ferris, J. J. , S. Kobayashi, and N. L.  Clesceri.  1974.  Growth of Selenastrum
     capricornutum in natural waters augmented with detergent products  in
     wastewaters.  Water Res. 8(12):1013-1020.

Filip, D. S., and R. I. Lynn.  1972.  Mercury accumulation by the  freshwater
     alga Selenastrum capricornutum.  Chemosphere. 6:251-254.

	, and E. J. Middlebrooks.   1975.  Evaluation of sample preparation
     techniques for algal bioassays.  Water Res. 9:581-585.

Fitzgerald, G. P.   1970.  Aerobic lake muds from the removal of phosphorus
     from lake waters.  Limnol. Oceanogr. 15(4):550-555.

	.   1971.  Comparative rates of phosphorus  sorption and  utili-
     zation by algae and aquatic weeds.  J. Phycol. Suppl 7, p.  11.

                   1972.  Bioassay analysis of nutrient availability,  p.  147-
     165 In. H. E. Allen and J. R. Kramer  [eds.], Nutrients in natural waters.
     John Wiley & Sons, Inc. , New York.

Forsberg, A.  1978.   Research on recovery  of polluted lakes:  Chemical and
     biological methods for monitoring wastewater effluent quality.  ACTA
     Univ.  Ups. Abstr. Upps. Diss. Sci. No. 458, 28 p.

Forsberg, C. G.  1972.  Algal assay procedure.  J. Water Pollut. Control Fed.
     44(8):1623-1628.

	, and A. Forsberg.  1972.  Algal growth potential  test improves
     sewage settlement control.  Ambio. 1(1):26-29.

     	, and 	.   1973.  Utslappskontroll vid Kommunala
     reningsverk:Minitest.  Vatten.  29(2):173-176.

     	, S. 0. Ryding, and A. Claesson.  1975.  Recovery of polluted
     lakes:  A Swedish research program on the effects of wastewater treatment
     and sewage diversion.  Water Res. 9(1):51-59.


                                       70

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Funk, W. H., F. W. Rabe, R. Filby, J.  I.  Parker, J.  E. Winner,  L.  Bartlett,  N.
     L. Savage, P. F. X. Dum'gan, Jr. ,  N. Thompson,  R. Condit,  P.  J.  Bennett,
     and K. Shaw.  1973.  Biological  impact  of  combined  metallic  and  organic
     pollution in the Cour d'Alene -  Spokane River drainage  system.   Washing-
     ton State Univ.  University of  Idaho.   OWRR (B-044  WASH and  B-015  IDA).
     187 p.

Gargas, E.  1973.  Preliminary statements on algal assay procedure at the
     Water Quality Research Institute,  Denmark,  p.  19-32 In Algal  assays in
     water pollution research.  Proc.  Nordic Symp.,  25-26 October 1972,  Oslo,
     Norway.  NORDFORSK, Secretariat  of Environmental  Sciences.

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.

               and R. G. Otto.  1976.   Algal  bioassays to evaluate a  proposed
     cooling lake.  J. Water Pollut. Control  Fed. 48(10):2351-2366.

Goldman, C. R. , M. G. Tunzi, and R. Armstrong.   1969.  Carbon-14  uptake as a
     sensitive measure of the growth of algal cultures,  p.  158-170  Ir\ E. J.
     Middlebrooks, T. E. Maloney, C. F. Powers,  and  L. M.  Kaack [eds.], Proc.
     of the Eutroph. Bioassess. Workshop,  19-21  June  1969.   U.S.  Pacific
     Northwest Water Laboratory, Corvallis Oregon.

Goldman, J. C.  1976.  Effects of temperature on growth constants of Selen-
     astrum capricornutum.  J. Water Pollut.  Control  Fed.  48(9):2215-2216.

               W. J. Oswald, and D. Jenkins.  1974.   The kinetics of inorganic
     carbon limited algal growth.  J. Water  Pollut. Control Fed. 46(3):554-
     574.

Greene, J. C., W. E. Miller, T.  Shiroyama, and E. Merwin.   1975.  Toxicity of
     zinc to the green alga Selenastrum capricornutum as a  function of phos-
     phorus or ionic strength,   p. 28-43  In  Proc. Biostim.  Nutr. Assess.
     Workshop, 16-17 October 1973.   U.S.  Environmental Protection Agency,
     Con/all is, Oregon.  EPA-660/3-75-034.   p. 28-43.

	, W. E. Miller, T.  Shiroyama and T.  E. Maloney.   1975.  Utiliza-
     tion of algal assays to assess  the effects  of municipal,  industrial, and
     agricultural wastewater effluents upon  phytoplankton production in  the
     Snake River system.  Water, Air and  Soil Pollut. 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, E. 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
     indigenous isolate Sphaerocystis to  predict effects of nutrient and zinc

                                      71

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     interactions upon phytoplankton growth in Long Lake, Washington.   Mitt.
     Int.  Ver.  Limnol. 21:372-384.

Haaland, P.  T.   1974.   Growth experiments with Selenastrum capricornutum.   J.
     Gen.  Microbiol.  8H2):13-14.

	,  and G.  Knutson.  1973.  Growth experiments with  Selenastrum
     capricornutum Printz.  p. 69-72 In Algal assays in water pollution re-
     search.  Proc. Nordic Symp., 25-26 October 1972, Oslo, Norway.   NORD-
     FORSK, Secretariat of Environmental Sciences.

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.

Harris, R.  F., E. J.   Brown, and J. F. Koonce.  1974.  Simultaneously  operating
     low and high affinity phosphate systems in aquatic microorganisms,   p.  80
     In K.  E. Marshall [ed.], XIX Cong. Internat. Assoc. Limnol.,  22-29 August
     1974,  Winnipeg,  Manitoba, Canada.

Hendricks,  A. C.  1978.   Response of Selenastrum capricornutum  to  zinc  sul-
     fides.  J. Water Pollut. Control Fed. 1:163-168.

Hostetter,  H. P.  1976.   A rapid bioassay for algal nutrients and  toxins.   J.
     Phycol. 12:10.

Hutchinson, T. C., and P. M.  Stokes.  1975.  Heavy metal toxicity  and algal
     bioassays.   p. 320-343 It) Water quality parameters.  Amer.  Soc.  Test.
     Mater., ASTM SIP 573.

Hwang,  C. P., T.  H. Lackie, and  P. M. Huang.  1976.  Absorption of inorganic
     phosphorus by lake  sediments.  J. Water Pollut. Control Fed.  48(12):2754-
     2760.

lonescu, A., and  L. Gavrila.  1972.  Contribution to study of the  influence  of
     some stimulatory and inhibitory substances on growth and photosynthesis
     on algae.   Stud. Cercet. Biol. Sci. Bot. 24(1):9-16.

Jadlocki, J. F.,  Jr., J.  Saldick, S. E. Coleridge, W. W. Smith, J.  W. Brown,
     and C. S.  Nicholson.  1976.   Effects of water hardness, phosphorus con-
     centration and sample pretreatment of the algal assay procedure—bottle
     test.  p.  323-334 In E. J. Middlebrooks, D. H. Falkenborg, and T.  E.
     Maloney [eds.],  Biostimulation and nutrient assessment.  Ann  Arbor Sci.

Katko, A.    1975.  Algal  assays for the  national eutrophication  survey,   p.  44-
     52 In  Proc. Biostim. Nutr. Assess. Workshop, 16-17 October 1973, U.S.
     Environmental Protection Agency, Corvallis, Oregon.  EPA-660/3-75-034.

Kallqvist,  T.  1973.   Algal assay procedure  (bottle test) at the Norwegian
     Institute for Water  Research,  p.  5-18  Iji Algal assays  in  water  pollution
     research.   Proc.  Nordic Symp., 25-26 October 1972, Oslo, Norway.   NORD-
     FORSK, Secretariat  of Environmental Sciences.

                                      72

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               1973.  Use of algal  assay  for  investigating a brackish water
     area.  p. 111-124 In Algal  assays  in water  pollution research.   Proc.
     Nordic. Symp., 25-26 October  1972,  Oslo,  Norway.   NORDFORSK,  Secretariat
     of Environmental Sciences.

	•   1974.  Algal growth potential  of  six  Norwegian waters receiving
     primary,  secondary and tertiary  sewage  effluents,   p.  100 lr\  K.  E.  Mar-
     shall  [ed.],  XIX Cong. Internat. Assoc.  Limnol.,  22-29 August 1974,
     Winnipeg, Manitoba, Canada.

Keznan, J.  D., and M. T. Auer.   1974.   The  influence of phosphorus luxury
     uptake on algal bioassays.  J. Water Pollut.  Control  Fed.  46(3):532-542.

King, D.  L., and J. T. Novak.   1974.  The kinetics of  inorganic carbon-limited
     algal  growth.  J. Water  Pollut.  Control  Fed.  46(7):1812-1816.

Klotz, R.  L., J. R. Cain, and F  R. Trainor.   1975.   A sensitive algal  assay:
     An improved method for analysis  of fresh waters.   J.  Phycol.  11(4):411-
     414.

Lee, C. C., R. F.  Harris, J.  K.  Syers,  and  D.  E.  Armstrong.   1971.   Adenosine
     triphosphate  content of  Selenastrum capricornutum.   Appl.  Microbiol.
     21 (5):957-958.

Lehmusluoto, P. 0.  1973.  Algal assay  procedure in  use in  Finland,   p.  33-34
     Irj Algal assays in water pollution research.  Proc.  Nordic Symp.,  25-26
     October 1972, Oslo, Norway.   NORDFORSK,  Secretariat of Environmental
     Sciences.

Lindmark,  G.  1973.  Bioassay with Selenastrum capricornutum to assess  the
     nutrient status of lakes and  the fertilizing influence of interstitial
     water,  p. 73-80 Irj Algal  assays in water pollution research.   Proc.
     Nordic Symp., 25-26 October 1972,  Oslo,  Norway.   NORDFORSK, Secretariat
     of Environmental Sciences.

Little, L., and D. Pittman.   1974.  Determination of herbicides and  their
     relationships to algal growth.   Proc.  Nebr.  Acad.  Sci.  Affil. Soc.  84:48.

Maki, A.  W., L. D. Geissel, and  H.  E. Johnson.   1975.   Toxicity of the  lampri-
     cide 3-trifluoromethyl-4-nitro phenol  to 10 species of algae,   p.  3-17  lr\
     U.S.  Fish Wildl. Serv. Invest. Fish Control  56.

Malone, R.  F.  , K.  A. Voos, W.  J. Grenney, and J.  H.  Reynolds.   1976.  The
     effects of media modifications upon Selenastrum capricornutum in batch
     cultures,  p. 267-292 I_n E. J. Middlebrooks,  D.  H.  Falkenborg,  and T.  E.
     Maloney [eds.], Biostimulation and nutrient assessment.   Ann  Arbor Sci.

Maloney,  T. E., W. E. Miller,  and  T.  Shiroyama.   1972.   Algal  responses to
     nutrient additions in natural  waters:   I. Laboratory assays,  p.  134-140
     Iji G.  F-  Likens [ed. ], Nutrients and eutrophication:   The limiting nu-
     trient controversy.  Spec.  Symp.   Vol.  I.,  Am.  Soc.  Limno.l. Oceanogr.


                                      73

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	5  w. E. Miller, and N. L. Blind.   1973.   Use  of algal  assays in
     studying eutrophication problems,  p. 205-214 ^n  S.  H.  Jenkins  [ed.],
     Advances in water pollution research.  Sixth Internat.  Conf., Jerusalem.
     Pergamon Press, Oxford and New York.

	,  and W. E. Miller.  1975.  Algal  assays:   Development and
     application,   p. 344-355 In Water quality parameters.   Amer.  Soc.  Test.
     Mater., ASTM STP 573.

Middlebrooks, E. J. , E.  A. Pearson, M. Tunzi, A. Adinarayana,  P.  H.  McGauhey,
     and G. A. Rolich.  1971.  Eutrophication of surface  water:   Lake Tahoe.
     J. Water Pollut. Cont. Fed. 43(2):242-251.

Miller, W. E. , and T. E. Maloney.  1971.  Effects of secondary and tertiary
     wastewater effluents on algal growth in a lake-river system.  J.  Water
     Pollut. Control Fed. 43(12):2361-2365.

                              and J. C. Greene.  1974.  Algal  productivity in
     49 lake waters as determined by algal assays.  Water  Res.  8:667-679.

	, J. C. Greene, T. Shiryoama, and E. Merwin.   1975.   The  use  of
     algal assays to determine effects of waste discharge  in  the  Spokane River
     system,  p. 113-131 Ij} Proc. Biostim. Nutr. Assess. Workshop,  16-17
     October 1973.   U.S. Environmental Protection Agency,  Corvallis, Oregon.
     EPA-660/3-75-034.

	, 	, and 	.   1976.  Application of  algal
     assays to define the effect of wastewater effluents upon algal  growth in
     multiple use river systems,  p. 77-92 Iji E. J. Middlebrooks,  D. A.  Fal-
     kenborg, and T. E. Maloney [eds.], Biostimulation and nutrient assess-
     ment.  Ann Arbor Sci.

	, 	, and 	.   1976.  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.

Mitchell, D.  1973.  Algal  bioassays for estimating the effect of added  mater-
     ials upon planktonic algae in surface waters,  p. 153-158 Jji E. Glass
     [ed.], Bioassay techniques and environmental chemistry.   Ann Arbor  Sci.

Monahan, T. J.   1973.   Lead inhibition among Chlorophycean micro  algae under
     phosphate deficient conditions.  J.  Phycol. Suppl. 9,  p.  13.

Morton, S. D.,  and T.  H. Lee.   1974.  Algal blooms:  possible effects  of iron.
     Environ.  Sci.  Technol.  8(7):673-674.

McDonald, G.  C., R.  D.  Spear,  P. J.  Lavin, and N. L. Clesceri.  1970.  Kin-
     etics of algal growth  in  austere media,   p. 97-105 In J.  E.  Zajic [ed.],
     Properties  and products of algae symposium.  Plenum Publ.  Corp.,  New
     York.
                                      74

-------
                 and N.  L.  Clesceri.   1973.   Effect  of wastewater  organic
     fractions on the growth  of  selected  algae,   p.  479-496  In  E.  Glass  [ed.],
     Bioassay techniques and  environmental  chemistry.   Ann Arbor  Sci.

Natarajan, K. V.  1959.  Mineral  nutrition  of  Selenastrum westii.   p.  156-161
     In P. Kachroo  [ed.],  Proceedings  of  the algology.   Ind.  Council Agri.
     Res. , New Delhi, India.

Oswald, W. J., and  S. A. Gaonkar.   1969.  Batch  assays  for determination  of
     algal growth potential,  p.  23-38 I_n E. J.  Middlebrooks, T.  E. Maloney,
     C. F. Powers,  and  L.  M.  Kaach  [eds.],  Proc.  of  the Eutroph.  Bioassess.
     Workshop, 19-21 June  1969,  U.S. Pacific Northwest  Water  Laboratory,
     Corvallis, Oregon.

Paoletti, C., B. Pushparaj, G. Florenzano,  P.  Capella,  and G. Lercker.  1976.
     Unsaponifiable matter of green  and blue-green algal lipids as  a factor of
     biochemical differentiation  of  their biomasses:  I. Total  Unsaponifiable
     and Hydrocarbon Fraction.   Lipids 11(4):258-265.

Parker, M.   1977.   The  use of algal  bioassays  to predict the  short and  long
     term changes in algal standing  crop  which result from altered phosphorus
     and nitrogen loadings.   Water  Res. 11:719-725.

Parr, M. P. , and R.  V.  Smith.  1976.   The identification of phosphorus as a
     growth-limiting nutrient in  Lough Neagh,  using  bioassays.  Water  Res.
     10:1151-1154.

Parra, B. 0.  1973.   Qualitative  study of phytoplankton of Loguna Verde,
     Concepcion (Chile), excluding  diatoms.  Gayana  Bot. 24:1-26.

Payne, A. G.  1973.   Environmental  testing  of  citrate bioassays for algal
     stimulation.   Proc. Conf. Great Lakes  Res.  16:100-115.

	.  1976.   Application  of  the algal  assay  procedure in  biostimula-
     tion and toxicity  testing,   p.  3-28  lr\ E. J. Middlebrooks, D.  H.  Falken-
     borg, and T. E. Maloney  [eds.], Biostimulation  and nutrient  assessment.
     Ann Arbor Sci.

	, and R.  H.  Hall.  1978.  A method  for measuring algal  toxicity and
     its application to the safety  assessment  of new chemicals.   Proc. Sec.
     Symp. Aquat. Tox., 31 October  and 1  November 1977,  Cleveland,  Ohio.  ASTM
     publ.   In press.

	, and 	.  1978.  Application of  algal assays  in the en-
     vironmental evaluation of new  detergent materials.  Mitt.  Int. Ver.
     Limnol.  In press.

Plumb, R. A. Sr.  1976.  A bioassay  dilution technique  to assess  the signif-
     icance  of dredged  material  disposal,   p.  335-346 I_n E. J.  Middlebrooks,
     D. H. Falkenborg,  and T. E.  Maloney  [eds.],  Biostimulation and nutrient
     assessment.  Ann Arbor Sci.


                                       75

-------
Plumb,  R.  H.  Jr., and G.  F.  Lee.   1974.  Phosphate algae and taconite  tailings
     in the western arm of Lake Superior.  Proc. Conf. Grt. Lakes  Res.  17(2):
     823-836.

Polesco-Ionasesco, L.  1974.   Biomass produced by green algae  Selenastrum
     bibraianum and Chlorella vulgaris: 157 cultivated in  laboratory and out
     of doors.   Lucr. Gradinii Bot.  Bureau.  1974:183-190.

Polescu, L.  1974.  Some physiological processes in the alga Selenastrum
     bibraianum.  An. Univ.  BuCur STUNT. Nat. 23:63-68.

Porcella, D.  B.  1969.  Continuous flow chemostat assays,  p.  7-22 T.TJ  E. J.
     Middlebrooks, T. E.  Maloney, C.  F. Powers, and L. M.  Kaach  [eds.], Proc.
     of the Eutroph.  Bioassess. Workshop, 19-21 June  1969, U.S.  Pacific North-
     west Water Laboratory,  Corvallis, Oregon.

               , P. A. Cowan,  and E.  J. Middlebrooks.  1973.  Biological re-
     sponse to detergent and nondetergent phosphorus in sewage:   Part  I.
     Water and Sewage Works 120(11):50-67.

	, 	,  and 	.  1973.  Biological  re-
     sponse to detergent and nondetergent phosphorus in sewage:   Part  II.
     Water and Sewage Works 120(12):36-46.

Rawlings, G.  D.  1978.  Source assessment:  Textile plant wastewater toxics
     study, Phase I.  Environmental Protection Technology Series  EPA-600/2-78-
     004.  Washington, D.C.  153 p.

Reynolds, J.  H.,  E.  J. Middlebrooks, D. B. Porcella, and W. J. Grenney.   1975.
     Effects of temperature on growth constants of Selenastrum capricornutum.
     J. Water Pollut. Control. Fed. 47(10):2420-2436.

	,  	, 	, and 	.   1975.
     Effects of temperature on oil refinery waste toxicity.  J. Water  Pollut.
     Control  Fed. 47(11):2674-2693.

	,  	, 	, and 	.   1976.
     Comparison of semi-continuous flow bioassays.  p.  241-266 In  E. J.  Mid-
     dlebrooks, D.  H. Falkenborg, and T.  E. Maloney  [eds.], Biostimulation  and
     nutrient assessment.  Ann Arbor Sci.

Rodhe, W.  1978.   Algae in culture and nature.  Mitt. Int. Ver.  Limnol.   In
     press.

Saldick, J.,  and J.  F. Jadlocki.  1978.   Solubi1ization of biological  avail-
     able phosphorus by autoclaving Selenastrum.  Mitt. Int. Ver.  Limnol.
     21:50-55.

Schelske, C.  L.,  E.  D. Rothman, and M. S.  Simmons.   1978.  Comparison  of bio-
     assay procedures for growth-1imiting nutrients  in the Laurentian  Great
     Lakes.  Mitt.  Int. Ver.  Limnol.   In  press.


                                       76

-------
Shiroyama, T., W. E. Miller, and J. C. Greene.   1975.   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.  En-
     vironmental Protection Agency, Corvallis,  Oregon.   EPA-660/3-75-034.

	» 	, and 	.   1976.   Comparison of the algal
     growth responses of Selenastrum capricornutum  Printz and  Anabaena  flos-
     aquae (Lyngb.) De Brebisson,  in waters collected from  Shagawa  Lake,
     Minnesota,  p. 127-148 In  E.  J. Middlebrooks,  D.  H. Falkenborg,  and T. E.
     Maloney  [eds.], Biostimulation and  nutrient assessment.   Ann Arbor Sci.

Skulberg, D. M.  1973.  A comparative  investigation of water from 15 European
     lakes,  p. 85-100 If} Algal assays in water pollution research.   Proc.
     Nordic Symp., 25-26 October 1972, Oslo,  Norway.   NORDFORSK,  Secretariat
     of Environmental Sciences.

                 1976.  Culture collection of algae.   Norwegian Inst. for
     Water Res. (NIWR) Q91, Blindren,  Norway.   9 p.

Smith, P. D.   1975.  The  use  of  in-situ  algal  assays  to evaluate the effects
     of sewage effluents  on the  production  of  Shagawa Lake phytoplankton.  p.
     143-173 In Proc. Biostim. Nutr. Assess. Workshop, 16-17 October 1973.
     U.S. Environmental Protection Agency,  Corvallis,  Oregon.   EPA-660/3-75-
     034.

Smock, L. A.,  D. L. Stoneburner, and J.  R.  Clark.   1976.  The toxic effects of
     trinitrotoluene (TNT) and its primary  degradation products on two species
     of algae  and the fathead minnow.  Water Res.  10(6): 537-543.

Smolen, M. D.  1975.  Prediction of algal growth potential from chemical
     nutrient  analysis,   p. 78 Jji V. W.  Langworthy [ed.], Proc. Third Ann.
     Pollut. Control Conf. of the Water  and Wastewater Equip. Manuf. Assoc.
     Ann Arbor Sci.

Soltero, R.  A., A. F- Gasperino, P. H. Williams, and  S. R. Thomas.  1975.
     Response  of the Spokane  River periphyton  community to primary sewage
     effluent  and contined investigation  of Long Lake.  D.O.E.  Project WF-6-
     75-081.   Completion  Report.  117  p.

	, D. M. Kruger, A. F. Gasperino,  J.  P.  Griffin, S. R. Thomas,
     and P.  H.  Williams.   1976.  Continual  investigation  of eutrophication in
     Long Lake, Washington.  Verification data for the Long Lake Model.
     D.O.E.  Project WF-6-75-081.  Completion Report.   64  p.

Steensland,  H.   1973.  Application of  the dialysis technique in testing  possi-
     ble growth retarding effects on algae  in  purified wastewater.  p. 47-49
     In Algal  assays in water pollution  research.   Proc.  Nordic Symp., 25-26
     October 1972, Oslo,  Norway.  NORDFORSK, Secretariat  of Environmental
     Sciences.

Steyn, D. J.,  D. F. Toerien, and J. H. Visser.   1974.  Continuous culture
     algal bioassays.  South Afr. J. Sci. 70(9):277-278.

                                       77

-------
Sturm,  R. N. , and A. G.  Payne.   1973.   Environmental  testing of trisodium
      nitrilotriacetate:   Bioassays  for  aquatic  safety and algal stimulation.
      p. 403-424  I_n  E. Glass  [ed. ],  Bioassay techniques and environmental
      chemistry.  Ann Arbor Sci.

Sugiki, A.   1977.   Algal  growth  potential  of tertiary effluents.   Japan Sewage
      Works  Agency.  Dept. of  Research and  Development (unpublished).

Thomas, N.  A., K. Hartwell, and  W.  E. Miller.   1975.   Great Lakes nutrient
      assessment,  p. 226-243  I_n  Proc. Biostim.  Nutr.  Assess.  Workshop, 16-17
      Octobar 1973,  U.S.  Environmental Protection  Agency,  Corvallis,  Oregon.
      EPA-660/3-75-034.

Thomas, R.  E., and  R. L.  Smith.   1975.   Assessing treatment process  efficiency
      with the algal assay test.   p.  244-248 _In  Proc.  Biostim.  Nutr.  Assess.
      Workshop, 16-17 October  1973,  U.S.  Environmental  Protection Agency,
      Corvallis,  Oregon.   EPA-660/3-75-034.

Toerien, D.  F.   1974.   Half saturation  constant for  nitrogen limited growth  of
      the green alga Selenastrum  capricornutum.  South Afr.  J.  Sci.  70(3):75-
      76.

	, and  C. H.  Huang.   1973.   Algal growth  prediction using growth
      kinetic constants.  Water  Res. 7(11):1673-1681.

	, and D. J. Steyn.   1974.   The  eutrophication  levels  of four
      South African impoundments,  p. 211  I_n  K.  E.  Marshall  [ed. ] ,  XIX Cong.
      Internat.  Assoc. Limnol.,  22-29 August  1974,  Winnipeg,  Manitoba, Canada.

	, K. L. Hyman, and M. J. Bruner.   1974.   Comparison of the algal
      bioassay responses of Selenastrum  capricornutum  and  Microcystis aejru-
      ginosa.  South Afr. J. Sci.  70(3):75.

Trainor, F. R. , and L. E. Shubert.  1973.  Growth  of  Dictyosphaerium, Selen-
      astrum, and Scenedesmus  (Chlorophyceae)  in  a  dilute  algal  medium.   Phyco-
      logia 12(l-2):35-39.

U.S.  Environmental Protection Agency.   1971.  Algal assay procedures:  Bottle
      test.   National Eutrophication Research  Program,  Corvallis, Oregon.   82 p.

U.S.  Environmental Protection Agency.   1974.  Biostimulation characteristics
      of wastes and receiving waters of  the Snake River basin.   National Field
      Investigations Center - Denver, Colorado and  Region  X,  Seattle, Wash-
      ington.  1974:33-37.

U.S. Geological  Survey.   1975.  Bioassay:  Algal growth potential  (AGP),   p.
      289-293 In P. E.  Greeson, T. A. Ehlke,  G. A Irwin, B.  W.  Lium, and K.  V.
      Slack [eds.], Methods for collection  and analysis of aquatic  biological
     and microbiological samples, Book  5,  Ch. A4.   Wash.  D.C.

Venkataraman, G.  S., and K. V. Natarrajan.   1958.   Molybdenum requirements  of
      Selenastrum westii   Current Sci.  27(11):454-456.

                                       78

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Vyas, H.  1975.  The effects of dyes  on  environment,   p.  251-269  In V.  W.
     Langworthy  [ed.], Proc. Third Ann.  Pollut.  Control  Conf.  of  the Water and
     Wastewater  Equip.  Manuf. Assoc.   Ann  Arbor  Sci.

Weiss, C. M.   1976.  Field  evaluation of the  algal  assay procedure on surface
     waters of North Carolina,  p. 29-76 In  E. J.  Middlebrooks, D.  H.  Fal-
     kenborg, and T. E. Maloney  [eds.],  Biostimulation and nutrient assess-
     ment.  Ann  Arbor Sci.

	.   1976.  Evaluation of the algal  assay procedure.   U.S.  Environ-
     mental Protection Agency, Corvallis,  Oregon.   Ecological  Research Series
     EPA-600/3-76-064.

	, and R. W. Helms.  1971.   Inter!aboratory precision test:   An
     eight laboratory evaluation  of  the  Provisional  Algal  Assay Procedure
     Bottle Test.   National  Eutrophication Research Program.   U.S.  Environ-
     mental Protection Agency, Corvallis,  Oregon.

Welch, E. B.   1976.  Eutrophication.   J. Water Pollut.  Control  Fed.  48(6):
     1335-1338.

Won, W.  D., L. H. DiSalvo,  and N. G.  James.   1976.   Toxicity  and  mutagenicity
     of  2,4,6-trinitrotoluene and its microbial  metabolites.   Appl.  Environ.
     Microbiol.  21(4):576-580.

Wright,  J. Jr.,  F.  A. Camp,  and J. Cairn,  Jr.  1974.   Preliminary algal  bio-
     assays to determine  nutrients limiting  algal  productivity in Mountain
     Lake, Virginia.  Assoc. SE Biol.  Bull.  21(2):92.
                                       79

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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

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          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

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     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


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                                              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

-------