&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-006
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.4500:
        Algal Toxicity

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                                     NOTICE

     This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number.  Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP)  in their title.

     The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also  used  by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this  guidance, the Agency uses the word "should."  In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline,  but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a  case-by-case basis.

     For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.qov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

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OCSPP 850.4500: Algal Toxicity.

(a) Scope—
       (1) Applicability.  This guideline is intended to be used to help develop data to submit to
       EPA under the Toxic Substances Control Act (TSCA)  (15 U.S.C.  2601,  et seq.), the
       Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.  136, et seq.), and
       the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).
       (2) Background.  The source material used in developing this  harmonized OCSPP test
       guideline includes the OPPT guideline under 40 CFR 797.1050 Algal Acute Toxicity
       Test; OPP 122-2 Growth and Reproduction of Aquatic Plants  (Tier 1) and OPP 123-2
       Growth and Reproduction of Aquatic Plants (Tier 2) (Pesticide Assessment Guidelines
       Subdivision J); Non-target Plants: Growth and Reproduction of Aquatic Plants  Tiers 1
       and 2 Standard Evaluation Procedure; OECD 201 Algal Growth Inhibition Test; ASTM E
       1218-04,  Standard Guide for Conducting Static 96-h Toxicity Tests with Microalgae; and
       OPP Pesticides Reregi strati on  Rejection Rate  Analysis:  Ecological Effects.   This
       guideline was formerly Public Draft OCSPP 850.5400 (April, 1996).

(b) Purpose. This guideline is intended for use in developing data on the toxicity of chemical
substances and mixtures ("test  chemicals" or "test substances") subject to environmental effects
test regulations.  This guideline prescribes test procedures and conditions designed to determine
the inhibition of growth and the pattern of growth in algae exposed to a test substance.  This
guideline was written specifically for the freshwater green alga Pseudokirchneriella subcapitata
(formerly known as Selenastrum capricornutum) and the marine diatom Skeletonema costatum
(see paragraph (e)(3)(i) of this guideline). Use of Navicula pelliculosa  or other test species may
require  some specific modifications in test procedures.  Methods for  toxicity testing with the
cyanobacterium species Anabaena flos-aquae (formerly classified as a blue-green alga) can be
found in OCSPP 850.4550. The Environmental Protection Agency will use data from these tests
in assessing the hazard and risks a test substance may present in the aquatic environment.  This
guideline  should be  used in  conjunction  with OCSPP 850.4000  (Background  and  special
considerations for conducting ecological  effects tests  with  terrestrial  and aquatic  plants,
cyanobacteria, and terrestrial soil core microcosms), which provides  general information and
overall  guidance for the  plant test  guidelines and OCSPP  850.1000  (Background and special
considerations for conducting ecological effects tests  with aquatic and  sediment-dwelling fauna
and aquatic microcosms), which provides general information for conducting toxicity tests in an
aqueous matrix.

(c) Definitions.  The definitions in OCSPP 850.1000 and OCSPP 850.4000 are applicable to this
guideline. In addition, the following more specific definitions also apply:

       Algicidal refers to having the property of killing algae.

       Algicidal concentration is the lowest concentration tested which allows no net growth of
       the population of test organisms during either exposure to the test substance or during the
       recovery period in the absence of test substance.

       Algistatic refers to having the property of inhibiting algal growth.


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       Algistatic concentration is the highest concentration tested which allows no net growth of
       the  population  of test organisms during exposure to the test substance but permits
       regrowth when the organisms are placed in test substance-free medium.

       Biomass is the dry weight of living matter present in a population and expressed in terms
       of a given volume, e.g. mg algae per liter.  Because  dry weight is difficult to measure
       accurately, surrogate measure of biomass, such as cell counts, are  typically used in this
       test.

       Growth refers to an increase in algal biomass in a specified period of time.

       Static system for this test refers  to a system in which old nutrient medium (test solution)
       is not renewed or replaced during the period of the test.

(d) General considerations—

       (1)  Summary  of the test.    Organisms  of a  particular species of  microalgae are
       maintained under static conditions in test vessels containing nutrient medium alone and
       nutrient medium to which the test substance has been added.  Over an exposure period of
       96 hours, data on population growth (cell density) are obtained on a daily basis.  The test
       is designed to determine the quantity of test substance that results in a 50 percent (50%)
       inhibition  (ICso) in biomass yield, growth rate, and the area under the growth curve.  The
       results of the test are expressed  as 96-hour median inhibition concentration (ICso) values.
       In addition, the no observed effect concentration (NOEC) for these response measures are
       determined.  The results are used to establish toxicity levels, evaluate hazards or risks to
       aquatic plants,  and to indicate if further testing at a higher tier is  necessary.   Note
       historically in OCSPP pesticide  and industrial chemical guidelines the term ECX was used
       to cover both the current OCSPP 850.4000 definition of EC* (concentration where x% of
       the  population exhibit the effect (e.g., mortality)) and 1C* (concentration resulting in a x%
       decrease or inhibition effect on an attribute of the population (e.g., growth rate)).

       (2)  General test guidance.  The general guidance in  OCSPP 850.4000  and OCSPP
       850.1000 applies to this guideline except as specifically noted herein.

       (3)  Range-finding test.   A range-finding  test is usually conducted to establish the
       appropriate test  substance solution concentrations for the definitive test.  In the range-
       finding test, the test organisms are exposed to a series of widely-spaced concentrations of
       the  test substance (e.g., 0.1, 1.0, 10, 100 milligrams per liter (mg/L), etc.). The details of
       the  range-finding test do not have to be the same as the definitive testing in that there are
       no replicates, and the number of test organisms used, and duration of exposure may be
       less than in definitive testing. In addition, the types and frequency  of observations made
       on test organisms are not as detailed or as frequently observed as that of a definitive test
       and results are analyzed using nominal concentrations. However, the  range-finding test
       will be more useful the  greater the similarity between the range-finding  test and the
       definitive test.

       (4)  Definitive  test.  The  goal  of the definitive test is to determine the concentration-
       response curves, 96-hour  ICso values (with 95% confidence intervals and standard error),

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       and NOEC and lowest observed effect concentration (LOEC) values for algal population
       growth based on yield,  growth rate, and area under the growth curve for each species
       tested.  The slopes of the concentration-response curves, and associated standard errors,
       and 95% confidence intervals of the slopes should be determined for all of the population
       growth  measures,  if  possible,  for each species.   However,  at a minimum, the full
       concentration-response curve (curve between  ICos to ICgo) is determined for the most
       sensitive measure of effect for a given species using a minimum of five concentrations of
       the test  chemical, plus appropriate controls. Recommend adding one or two additional
       test concentrations in the lower tail of the concentration-response curve  for the most
       sensitive endpoint to ensure bracketing of the most sensitive NOEC or ICos value. For a
       satisfactory test for a given species, the lowest treatment concentration is below the yield,
       average specific growth rate, and area under the growth curve for all ICso values and is at
       or below the NOEC (or ICos) for all growth measures.  Analytical confirmation of test
       concentrations is performed  as  described  in  OCSPP 850.1000.   A  summary of test
       conditions is provided in Table 3 of this guideline and validity elements for an acceptable
       definitive test are listed in Table 4 of this guideline.

       (5) Limit test.  In some situations, it is only necessary to ascertain that both the 96-hour
       ICso values for growth measures are above a certain limit concentration,  and that at this
       limit concentration there is no observable  adverse effect on growth.   For pesticides, a
       limit test has also been referred to as a Tier I test or Maximum Challenge Concentration
       test. In an algal limit test, at least four replicate test vessels are exposed to a single "limit
       concentration,"  with the same number of test vessels  containing the appropriate control
       solution(s).  The multiple-concentration  definitive test may be waived for a given test
       species if the following two conditions are met for yield, average specific growth rate and
       area under the growth curve.  First, the "limit" treatment response is statistically less than
       a 50% decrease from the  control response (i.e.., ICso value > limit concentration), and
       second,  the limit treatment responses are not significantly reduced  (or inhibited) as
       compared tithe control response (i.e., NOEC > limit concentration). For  most industrial
       chemicals,  1,000 mg/L or the limits of water solubility or dispersion  are considered
       appropriate  as  the limit  concentration.   For pesticides,  the  limit  concentration is
       equivalent to the maximum label rate (pounds of active ingredient per acre (Ibs a.i./A))
       directly  applied to a one acre pool that is 6 inches deep (21,280  cubic feet (ft3) or 602,581
       liters).  For example, a  1 Ib a.i./A (or 453,592 milligrams (mg) a.i. per acre) application
       rate  and assuming a water density  of 1 gram  per milliliter  would  have  a  limit
       concentration of 0.75  mg a.i./L.   Except for the  number  of treatment groups,  an
       acceptable limit test follows the  same test procedures, is the same duration, and has the
       same number of controls as the multi-concentration definitive test (Table 3). Acceptable
       limit  tests  like  definitive   tests include  analytical  confirmation  of  the  exposure
       concentration.

(e) Test standards—

       (1) Test substance.  For industrial chemicals, the  substance to be  tested should be
       technical grade unless the test is  designed to test a specific formulation, mixture, or end-
       use product. For pesticides, the  use of the typical end-use product (TEP) instead of the
       technical grade active ingredient (TGAI) is preferred  for all aquatic plant phytotoxicity


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tests.  If there is more than one TEP with the same inert substances, the one with the
highest percent a.i. and/or the one most commonly used should be tested.  If there is more
than one TEP with different inert substances, a TEP representative of each different inert
substance should be tested in the range-finding test and at a minimum the most sensitive
one tested in the definitive or limit test.   Adjuvants are not used with TEP or  TGAI
testing of algae.  OCSPP 850.1000 and OCSPP 850.4000 list the type of information that
should be known about the  test substance before testing,  and discusses methods for
preparation of test substances.

(2) Test duration. The test duration is 96 hours.

(3) Test organism—

       (i) Species.

              (A) This test  guideline prescribes testing conditions and standards for
              three  algal  species:  the  freshwater  green  alga  Pseudokirchneriella
              subcapitata (formerly known as Selenastrum capricornutum and also as
              Raphidocelis subcapitata); the marine diatom, Skeletonema costatum; and
              the freshwater diatom, Navicula pelliculosa.   For  pesticides all three of
              these  species  are tested  at a minimum.   These  species  have  been
              historically used for this type of testing. For testing industrial  chemicals,
              the specific algal species tested are selected on a case-by-case basis.

              (B) Some species that may be tested in addition include Desmodesmus
              subspicatus (formerly Scenedesmus subspicatus), Microcystis aemgmosa,
              Thalassiosira  pseudonana,  Dunaliella tertiolecta  and  Phaeodactylum
              tricornutum.  Appropriate references (as given in paragraph (j)(3),  GX7),
              GX8X (j)(9), (j)(10), and G)(15) of this guideline) should be consulted
              regarding the correct culturing and testing conditions for these species.

              (C) Procedures  for testing the  cyanobacterium, Anabaena flos-aquae,
              formerly classified as a blue-green alga, are found in OCSPP 850.4550.

       (ii) Source, age and condition.

              (A) All algae used for a particular test should be from the same source and
              the same stock culture. Also, the clone of all  species should be specified.
              Algae to  be  used  in toxicity  tests  may  be  initially  obtained  from
              commercial  sources  and  subsequently cultured  using sterile  technique.
              Upon receipt of a culture of a species not previously maintained  in the
              testing facility,  a period of six weeks  culturing is recommended  to
              establish the ability to successfully maintain a healthy,  reproducible-
              growing culture.  The algal inoculum to begin the toxicity test should be
              from logarithmically-growing stock cultures (typically 3- to 7-days old).
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              (B) A culture should not be used for starting a test under the following
              conditions:

                     (1) If it is not in the log growth phase;

                     (2) If visual examination at 400 power shows it is contaminated by
                     fungi or other algae, or if the health of the culture is doubtful in
                     any respect;

                     (3) If test algae were used in a previous test, either in a treatment
                     or a control.

       (iii) Culturing procedures.

              (A) Algal  cultures  should  be maintained in  freshwater or  saltwater
              medium  as described in  the references listed in paragraphs Q)(l),  (J)(3),
              (j)(6), and  (j)(9) of  this guideline.  The cultures should be maintained
              under the same conditions as used for testing.

              (B) Aseptic stock transfer should be performed on a regular schedule (e.g.,
              once or  twice weekly)  to  maintain a  supply  of cells in or  near the
              logarithmic growth phase.  Long-term maintenance of cultures on a solid
              medium  containing 1% agar in sterile Petri plates or test tubes may be
              desirable.  However for a satisfactory test, the  algal inoculum used to
              initiate toxicity testing is  from  a  liquid culture shown to be  actively
              growing  (i.e. capable of logarithmic growth within the test period) in at
              least two subcultures lasting 7 days each prior to the start of the definitive
              test.

              (C) Stock algal cultures of P.  subcapitata  and N. pelliculosa should be
              shaken on  a rotary  shaking  apparatus.  Culture vessels containing S.
              costatum should be shaken by hand once or twice daily.  If clumping of
              cells is not  experienced, S. costatum  may be  continuously shaken at
              approximately 60 cycles per minute.

(4) Administration of test substance—

       (i) Preparation of test solutions—

              (A) Stock solutions  or direct addition.  Test solutions are prepared by
              adding the  test substance to the nutrient medium either  as direct addition
              or by addition of a stock solution.  Typically, a stock solution of the test
              substance is prepared and aliquots of the stock solution  added to medium
              to obtain the desired  test concentrations.  Guidance for preparation of test
              solutions, especially  for difficult or low  solubility test  substances, is
              provided in OCSPP 850.1000.
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       (B) Solvents.  The recommended solvent for algal toxicity tests is N,N-
       dimethyl-formamide, as solvents such as acetone can cause stimulation of
       bacterial growth.   The concentration of solvent should preferably  be the
       same in all test treatments and should not exceed 0.1 milliliters per liter
       (mL/L).

       (C) Stock solution pH adjustment.   The pH may be adjusted in stock
       solutions to match the pH of the medium if pH change does not affect the
       stability  of  the  test  substance in  the stock  solution  or test solution.
       Hydrochloric acid (HC1) and  sodium hydroxide (NaOH) may be used for
       this adjustment if warranted.  The pH should  not be  adjusted after the
       addition of the test substance or stock solution into the test medium  except
       as discussed in paragraph (e)(9)(iv)(C)f2y) of this guideline.

       (D) Exposure technique.

              (1) This  test  is  conducted using a static exposure  technique.
              Although semi-continuous algal culturing techniques are available,
              they  have not  been commonly employed  in algal  toxicity  testing
              and their use is not required.

              (2) When a substance is known to have a tendency to vaporize,
              closed  test flasks with  increased  head-space   may  be  used.
              Attempts should be made to determine the amount of the substance
              which remains in  solution, and extreme caution is advised when
              interpreting results of test with volatile chemicals using  closed
              systems.

(ii) Treatment levels.  For a  given plant species a minimum of five test solution
concentrations  are  tested for  multi-concentration definitive  testing, plus  the
appropriate  control(s).    A  range-finding test can be  used  to establish  the
appropriate test solution  concentrations for the  definitive test  (see paragraph
(d)(3)  of this  guideline).  For  scientifically  sound estimates of a given point
estimate (e.g.,  ICso,  ICos) test  substance  concentrations should immediately
bracket the point  estimate(s)  of concern.   The  concentrations should be a
geometric progression of twofold at  a minimum (e.g., 0.1, 0.2, 0.4, 0.8, and 1.6
milligrams per liter (mg/L)).  While a twofold progression is preferred, threefold
and  fourfold  progressions are  acceptable.    If a fourfold  or higher   series
progression is  used,  the  rationale  for  using this large an interval  between
concentrations and  the  effect on the accuracy and reproducibility of the point
estimate(s) and NOEC should be provided.  For an acceptable study, the  lowest
test treatment level should be lower than the ICso  values for yield and average
specific growth  rate based on cell density.   The lack of a NOEC for an effect
measure is not critical as long  as the response-curve for the effect measure is
acceptable  for  calculation of  the 5% inhibition  concentration  (ICos).   It is
recommend that one or two additional test concentrations in the lower tail of the
concentration-response curve  of the most sensitive endpoint for a given species be
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       added to ensure bracketing of both the most sensitive ICso value and the most
       sensitive NOEC  (or ICos) value. For a  limit test,  there is single treatment
       concentration,  plus  the  appropriate  control(s).     Guidance  on  the  limit
       concentration is provided in paragraph (d)(5) of this guideline.

       (iii) Introduction of test organisms.

              (A) In preparation for the test, appropriate volumes of nutrient medium
              and/or test solution are placed in the test vessels. Within 30 minutes algae
              are then introduced  into the test vessels, which  are subsequently placed in
              a growth  chamber  or a laboratory testing area.  Inoculum  should be
              impartially or randomly distributed  among  the test  vessels  in such  a
              manner that test results show no significant bias from the distributions.

              (B) Test vessels within the testing area are positioned in a random manner
              or in a way in which appropriate  statistical  analyses can be used to
              determine the variation due to placement. Because illumination may vary
              at different positions within the growth chamber, and since this parameter
              has an important influence on algal growth, it is recommended that the test
              vessels be randomly re-positioned each day.

(5) Controls.

       (i) Every test includes a negative control consisting of the same nutrient medium,
       conditions, procedures, and algae from the same culture, except that none of the
       test substance is added.  In  addition, vehicle (solvent) controls are  also included if
       a solvent is used to dissolve or suspend the test substance.

       (ii) For a satisfactory test,  cell counts in the  controls should increase by a factor
       of at least 100 times for P. subcapitata  and a factor of at least  30  times for S.
       costatum by test termination (i.e., logarithmic growth in the controls).

       (iii) At test termination the coefficient of variation for  mean control yield should
       be less than 15% and it should be less than 15% for average specific growth rate,
       which is a logarithmically-transformed variable.

(6) Inoculum concentration and replicates.  The  minimum number of replicates per
treatment and control  is four to provide acceptable confidence in the  results and the
ability to conduct the statistical hypothesis tests.  Each test vessel should be inoculated at
an initial population density to allow  sufficient growth under the  test conditions to
provide accurate quantification without resulting in nutrient or carbon dioxide limitation.
Recommended initial  population  density is 10,000 cells/mL for Pseudokirchneriella
subcapitata (formerly Selenastrum capricornutum)  and Skeletonema costatum.  These
inoculum concentrations are known to result in acceptable population densities at the end
of 96 hours under the specified  test parameters for these species.  The use  of other test
species such as Navicula pelliculosa may require higher initial inoculum  concentrations,
and the initial population density  necessary to result in sufficient growth over the  test
period should be determined on  a case-by-case basis. In no case should a test be started

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with less than 10,000 cells/mL. Each test vessel should contain an equal volume of test
solution  and the same initial inoculum volume and  concentration.   The volume of
inoculum used should not  dilute the test  solution (i.e.., should typically be less than 2
mL).  The volume of inoculum to be  added to each vessel is calculated based upon the
cell concentration in the stock solution, the volume in the test vessel, and the desired
initial cell concentration.

(7) Facilities, apparatus and supplies. Normal laboratory equipment and especially the
following are necessary:

       (i) Containers for  culturing  and testing algae.  Erlenmeyer flasks should be
       used for test vessels.  The flasks may be of any volume between 125 and  500
       milliliters (mL) as long as the same size  is  used throughout a test and the  test
       solution  volume  does  not exceed 50%  of  the flask volume.   To permit  gas
       exchange but  prevent contamination, the flasks  should be covered with foam
       plugs,  stainless  steel caps,  Shimadzu enclosures,  glass  caps or  screw caps.
       Containers and covers that may contact the test solution  should be chosen to
       minimize sorption of test substances, and not contain substances  that can be
       leached or dissolved into aqueous  solutions  in quantities that can affect the  test
       results.   The acceptability  of foam  plugs should be investigated  prior to  use
       because some brands have been found to  be toxic.  For a satisfactory test for a
       given test species, all test vessels and covers in the test are identical.

       (ii) Growth  chamber or laboratory environment.

              (A) A growth chamber or a laboratory environment that can  hold the  test
              vessels and  will  maintain  the air temperature, lighting  intensity,  and
              photoperiod  specified in this test guideline. If necessary for the given  test
              species, a  mechanism for continuously shaking the test  vessels should be
              incorporated into the growth chamber  or controlled environment room.

              (B) Facilities should be well ventilated and free of fumes that may affect
              the test organisms.   Construction materials  and  equipment that may
              contact the stock  solution,  test solution, or nutrient medium should be
              chosen to minimize sorption of test substances and not contain substances
              that can be leached or dissolved into aqueous solutions in quantities that
              can affect the test results.   Refer to OCSPP 850.1000  for additional
              information on appropriate construction materials.

       (iii) Environmental monitoring  equipment.   Equipment  for determination of
       test conditions (e.g.., pH  meter, photosynthetically active radiation  (PAR) light
       sensor, etc.)

       (iv) Cleaning and sterilization.  Apparatus  for preparing sterile nutrient media.
       Apparatus for sterilizing glassware  and maintaining aseptic  technique during
       culturing and testing. New test vessels may contain substances that inhibit growth
       of algae.  They are  therefore to be cleaned thoroughly and used several times to
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       culture algae before being used in toxicity testing.  All glassware used in algal
       culturing or testing is to be cleaned and sterilized prior to use.

       (v) Microscope. Microscope capable of 100 to 400 power magnification.

       (vi) Algal cell counting equipment.  An apparatus for enumerating algae,  e.g.,
       hemacytometer, plankton counting chamber, or electronic  particle counter.   An
       alternative method to performing cell counts is to determine the chlorophyll a
       concentration through spectrophotometric or fluorometric methods, as described
       in the reference in paragraph (j)0) and (j)(6) of this guideline.

       (vii) Nutrient media.

              (A) Water used for preparation of nutrient medium should be of reagent
              quality (e.g., ASTM Type I water).  Freshwater algal nutrient medium is
              prepared by  adding  specified  amounts of reagent-grade chemicals  to
              reagent  water.  Marine  algal nutrient medium is prepared  by adding
              reagent  grade  chemicals  to  synthetic salt water or filtered  natural salt
              water,  or  by  preparing  a complete  saltwater medium.   Salinity  for
              saltwater medium should be 30 parts per thousand (ppt) ± 5 ppt.

              (B) Formulation and sterilization of nutrient medium used for algal culture
              and  preparation  of test  solutions should conform  to those currently
              recommended  by  ASTM for freshwater and  marine algal toxicity tests
              described in the reference in paragraph (j)(l) of this guideline and Tables
              1  and 2.  Chelating agents (e.g. ethylenediaminetetraacetic acid (EDTA))
              are included in the nutrient medium for optimum cell growth.  Lower
              concentrations of  chelating agents,  down to one-third  of  the  normal
              concentration recommended for AAP medium, may be used in the nutrient
              medium used  for  test solution preparation if it is  suspected that  the
              chelater will interact with the test substance.  Nutrient medium should be
              freshly prepared for algal testing or may be stored under refrigeration in
              the dark at 4 degrees Celsius  (°C).  Nutrient medium  should be sterilized
              by autoclaving or filtering (0.22 micrometer (|im) membrane filter).  At
              the start of the test, the pH of the nutrient medium should be 7.5 ±0.1 for
              freshwater algal medium and 8.0 ±0.1 for marine algal medium. The pH
              may be adjusted prior to addition of the test substance with 0.1 normal (TV)
              or IN sodium hydroxide (NaOH) or hydrochloric acid (HC1).

(8) Environmental conditions.   Environmental conditions during the test should be
maintained as specified below:

       (i) Temperature.  The test  temperature is 24 °C for P. subcapitata  and N.
       pelliculosa, and 20 °C for S. costatum.  Excursions from the test temperature
       should be no  greater than ± 2 °C.  Temperature monitoring is  described  in
       paragraph (e)(9)(iv)(A) of this guideline.
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              (ii) Lighting  and photoperiod.  Test vessels containing P. subcapitata and N.
              pelliculosa  are illuminated  continuously;  those  containing  S. costatum are
              provided a 14-hour light/10-hour dark photoperiod.  Cool-white fluorescent lights
              providing 60 micromoles per square meter per second (|imo/m /s), for cool-white
              fluorescent  lighting this is  approximately  equivalent to 4300 lux, are used.
              Additional information on the use of lighting in plant toxicity tests can be found
              in the references given in OCSPP 850.4000.

              (iii) Shaking.  Test vessels containing these species should be placed on a rotary
              shaking apparatus and oscillated at approximately 100 cycles/min during the test.
              The rate of oscillation should be determined  at the beginning of the test or at least
              once daily during testing if the  shaking rate is changed or changes.  Test vessels
              containing S. costatum should be shaken by hand once or twice daily. If clumping
              of  cells  is  not   experienced,  S.  costatum may  be  continuously shaken  at
              approximately 60 cycles per minute.
                  Table 1.—Preparation of Medium for Freshwater Algae
This medium (referred to as AAP medium) is prepared by adding 1 milliliter (ml) of each of the
macronutrient stock solutions and 1 ml of the micronutrient stock solution listed below to approximately
900 ml reagent grade water and then diluting to 1 liter (L).	
Macronutrient stock solutions are made by dissolving the following into 500 ml of reagent grade water:
  Sodium nitrate (NaNO3) -12.750 grams (g)
  Magnesium chloride hexahydrate (MgCI2 • 6H2O) - 6.082 g
  Calcium chloride dihydrate (CaCI2 • 2H2O) - 2.205 g
  Magnesium sulfate heptahydrate (MgSO4 • 7H2O) - 7.350 g
  Potassium phosphate (K2HPO4) - 0.522 g
  Sodium bicarbonate (NaHCO3) -  7.500 g	
The micronutrient stock solution is made by dissolving the following into 500 ml of reagent grade water:
  Boric acid (H3BO3) - 92.760 milligrams (mg)
  Manganese chloride tetrathydrate (MnCI2 • 4H2O) - 207.690 mg
  Zinc chloride (ZnCI2) -1.635 mg
  Ferric chloride hexahydrate (FeCI3 • 6H2O) - 79.880 mg
  Cobalt chloride hexahydrate (CoCI2 • 6H2O) - 0.714 mg
  Sodium molybdate dihydrate (Na2MoO4 • 2H2O) - 3.630 mg
  Copper chloride dihydrate (CuCI2 • 2H2O) - 0.006 mg [Typically must be prepared by serial dilution].
  Ethylenediaminetetraacetic acid disodium salt dihydrate  (Na2EDTA • 2H2O) -150.000 mg
  Sodium selenite pentahydrate (Na2SeO3 • 5H2O) - 0.005 mg [Used only in medium for stock cultures of
diatom species]	
For freshwater diatom species only, add sodium metasilicate nonahydrate (Na2SiO3 • 9H2O) as another
macronutrient.  May be added directly (202.4 mg) or by way of stock solution to give 20 mg/L silicon (Si)
final concentration in medium (see reference in paragraph (j)(1) of this guideline).	
Adjust pH to 7.5 ±0.1 with 0.1 N or 1.0 N sodium hydroxide (NaOH) or hydrochloric acid (HCI).
Filter the media into a sterile container through a 0.22 micrometer (urn) membrane filter. Store medium in
the dark at approximately 4 degrees Celsius (°C) until use.	
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                   Table 2.—Preparation of Medium for Saltwater Algae
This medium (referred to as MAA medium) is prepared by adding the aliquots of Metal Mix, Minor Salt Mix
and Vitamin Mix listed below into a sterile container containing approximately 900 milliliters of natural or
artificial salt water (salinity 30 ± 5 ppt) then diluting to 1 L. Either natural salt water that has been filtered
through a 0.22 urn membrane filter or reconstituted salt water is used.

Add the amounts given below to prepare medium used fortoxicity testing. Add twice the amounts given
to prepare medium for use in maintenance of stock cultures.
Add 15 ml of Metal Mix
Add 10 ml of Minor Salt Mix stock solution
Add 0.5 ml of Vitamin Mix stock solution (Add 1 ml of vitamin mix if Thalassiosira is used).	
The Metal Mix is prepared by adding the specified amount of chemicals in the order listed below to 900
milliliters (ml) reagent water and diluting to 1 liter (L).
Metal Mix:
Ferric chloride hydrate (FeCI3 • H2O) - 0.048 g
Manganese chloride tetrathydrate (MnCI2 • 4H2O) - 0.144 g
Zinc sulfate heptahydrate (ZnSO4 • 7H2O) - 0.045 g
Copper sulfate pentahydrate (CuSO4 • 5H2O) - 0.157 mg
Cobalt chloride hexahydrate (CoCI2 • 6H2O) - 0.404 mg
Boric acid (H3BO3)-1.140 g
Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA • 2H2O) -1.0 g	
The Minor Salt Mix is prepared by adding the specified amounts of the chemicals listed below to 900 ml
reagent water and diluting to 1 L.
Minor Salt Mix:
Potassium phosphate (K3PO4) - 0.3 g
Sodium nitrate (NaNO3) - 5.0 g
Sodium metasilicate nonahydrate (NaSiO3 • 9H2O) - 2.0 g	
The Vitamin Mix is prepared by adding the specified amount of chemicals in the order listed below to 900
ml reagent water and diluting to  1 L.
Vitamin Mix:
Thiamine hydrochloride - 500 mg
Biotin -1  mg
cyanocobalamin (vitamin B12) -1.0 mg	
Adjust MAA medium pH to 8.0 ± 0.1 with 0.1 N or 1.0 N NaOH or HCI. Store medium in the dark at
approximately 4 °C until use.	
       (9) Observations—
              (i) Measurement of test substance.  Analytical confirmation of dissolved test
              concentrations is performed at a minimum at test initiation and at test termination
              for static tests, as described in OCSPP 850.1000.  The analytical methods used to
              measure the amount of test substance in a sample are validated before beginning
              the test, as described  in  OCSPP 850.1000.   Samples  for  analysis  should be
              collected as described in OCSPP  850.1000, with the following exception:  at the
              end of the test and after aliquots have been removed for algal  growth-response
              determinations,  microscopic  examination,  mortal  staining,  or  subculturing, the
              replicate test containers for each chemical concentration  may be pooled into one
              sample.  An aliquot of the pooled sample  may then be  taken and the dissolved
              concentration of test substance in solution determined after all algal  cells have
              been removed, either by centrifugation or filtration.  The effect of centrifugation
              or filtration upon recovery of the test  substance should be determined during
              method validation.  As  an additional procedure, the mass of test substance sorbed


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to the algae may be determined. To measure sorbed test substance, separate and
concentrate the algal cells from the remaining pooled sample by centrifuging or
filtering  and  measure  the  test   substance  concentration  in  the  algal-cell
concentrate.

(ii)  Test solution appearance.  Observations are made daily on test substance
solubility (e.g., surface slicks,  clarity, precipitates, or material adhering to the
sides of the test vessels) and recorded.

(iii) Dilution water quality.  The  dilution water source used to  prepare media
should be  periodically analyzed to  document  and characterize  the  hardness,
alkalinity,  pH,  conductivity,  total  organic carbon  (TOC)  or chemical oxygen
demand magnitude and variability, and to ensure that pesticides, PCBs  and toxic
metals are  not present at concentrations  that are considered toxic.  See OCSPP
850.1000 for guidance on dilution water.

(iv) Environmental conditions—

       (A) Temperature.  It is impractical to measure the  temperature of the
       solutions  in  the  test  vessels  while  maintaining  axenic  conditions.
       Therefore, one or two extra test vessels may be prepared for the purpose of
       measuring the solution temperature during the test.  Alternatively, hourly
       measurements of the  air temperature  (or  daily measurements  of the
       maximum and  minimum) are  acceptable.  Because the test vessels are
       placed in an environmental  chamber or incubator, the air  temperature is
       more likely to fluctuate than the water temperature.

       (B) Light intensity.  Light intensity should be monitored at test initiation
       at the approximate level of the test solution at each test vessel position in
       the  growth chamber or environmental control room.   If it is suspected
       during  the test that  light  intensity  has  changed by 15%  or more,
       monitoring  of  light   intensity   should  be   conducted   daily.     A
       photosynthetically active radiation (PAR) sensor is used to measure light
       quality.  The light intensity  should not vary more than ±  15% from the
       selected light  intensity  at any test vessel position in the  incubator or
       growth chamber.  Because illumination may vary at  different positions,
       and since this  environmental  parameter has an important influence on
       growth, it is  recommended that the test vessels be randomly re-positioned
       on a regular schedule (e.g.., daily) to minimize spatial differences.
       (C) pH.
              (1)  The pH of all test solutions is measured at test initiation and
              termination.   This measurement may be made on the  bulk test
              solutions at test initiation and on  samples of pooled replicates of
              each test  treatment  at test termination provided none  of the
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              replicates appear to be "outliers" with respect to growth, in which
              case individual pH measurements should be made.

              (2) Before addition of the inoculum, if the test substance is highly
              acidic and reduces the pH of the test solution below 5.0 at the first
              measurement, or is highly basic and increases the pH of the test
              solution  similarly,  appropriate  adjustments to  pH  should be
              considered  and the test solution measured for pH on each  day of
              the test.

(v) Measures of effect—

       (A) Cell count. At the end of 24, 48, 72, and 96 hours, at a minimum the
       algal cell density (cells per milliliter) in all  test vessels are enumerated by
       performing cell counts using direct microscopic observation or using an
       electronic particle counter.    Additional  confirmation  measurements
       include   chlorophyll    a    (measured    spectrophotometrically   or
       fluorometrically).

              (1) Microscopic counting. Microscopic counting of cells  can be
              performed using a hemacytometer or an inverted microscope with
              settling chambers.  Precision is proportional to  the square root of
              the number of cells counted.   For  microscopic counting,  two
              samples should be taken from each  test vessel and two  counts
              made of each sample. Whenever feasible, at least 400 cells per test
              vessel should be counted in order to obtain ± 10% accuracy at the
              95% confidence level.

              (2) Electronic  particle  counter.    An alternative method to
              enumerate  large  numbers  of cells very rapidly is to  use an
              electronic  particle counter.   To use this method,  an electronic
              particle count for a sample is converted to a cell count using data
              developed by the laboratory demonstrating the correlation between
              electronic  particle  counts and microscopic counts for each algal
              test species. Automated particle counting, although the most rapid
              and sensitive method,  has limitations, some related to particle
              interferences. If the test solution does not have a low background
              in the particle  size range of the test species, masking errors will
              result.  An additional test vessel at  each concentration containing
              test substance  and  growth  medium  but no  algae  can allow
              measurement of, and if needed, correction for, potential particle
              interference.

       (B) Appearance  and  condition.  Note daily any unusual cell  shapes,
       color differences,  differences  in chloroplast morphology,  flocculations,
       adherence of algae to test vessels, or aggregation of algal cells.   These
       observations are qualitative and descriptive, and are not used in endpoint
                        Page 13 of 26

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calculations.  They can be useful in determining additional effects of test
substances.  In addition, microscopic observations at test termination are
performed to  determine whether the altered  growth response  between
controls   and   test  algae  (at  the  concentrations  of  test  substance
demonstrating an effect) was due to a change in relative cell numbers, cell
sizes, or both.

(C) Algistatic and algicidal determination. At the end of the definitive
test, determination of algistatic and algicidal effects may be performed.
When performed  algistatic effects may be differentiated from  algicidal
effects by  either  Method  1  or  Method 2   described  in paragraphs
(e)(9)(v)(C)(7;  and (e)(9)(v)(C)(2; of this guideline, respectively.

       (1) Method 1.  Add 0.5 mL of a 0.1% solution (weight/volume) of
       Evans blue stain to a  1-mL  aliquot of algal suspension from a
       control  vessel and to a  1-mL aliquot of algae from the test vessel
       having the lowest concentration of test substance which completely
       inhibited  algal  growth.  If  algal  growth was  not completely
       inhibited, select an aliquot of algae for staining from the test vessel
       having  the  highest concentration of test substance that inhibited
       algal growth.   Wait 10 to 30 minutes,  examine microscopically,
       and determine  the percent of the cells  that stain  blue (indicating
       cell mortality).  A staining control is also performed concurrently
       using heat-killed or formaldehyde-preserved algal  cells;  100% of
       these  cells should stain  blue.   This  method  will  work  for S.
       costatum and possibly Navicula spp., but probably will not work
       with P.  subcapitata.

       (2) Method 2.  Remove 0.5 mL aliquots of test solution containing
       growth-inhibited algae from each replicate test vessel having the
       lowest  concentration of test substance  that completely inhibited
       algal growth.  If algal growth was not completely inhibited, select
       aliquots from  the  highest  concentration of test  substance  that
       inhibited algal  growth.   Combine these  aliquots  into a  new test
       vessel and add  a  sufficient volume of fresh nutrient medium to
       dilute the  test  substance  to  a concentration that does not affect
       growth  (using the original test vessel size and solution volume is
       generally appropriate).  Aliquots from the control test vessels are
       also transferred to clean medium. Incubate these subcultures under
       the environmental conditions used in the definitive test for a period
       of up to 9 days, and observe periodically (e.g., every other day) for
       algal  growth  (direct or  indirect  methods) to  determine  if  the
       algistatic effect noted after the 96-hour definitive exposure test is
       reversible.  This subculture test may be discontinued as soon as
       growth  occurs.
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(f) Treatment of results—

       (1)  Response  variable  calculation.    Algal  population  density  is  the  biomass
       measurement normally used in this test  guideline  to evaluate  the  inhibitory  and
       stimulatory effects of the test substance.  Three response variables  are calculated from
       algal density: final population density, also referred to as yield, as described in paragraph
       (f)(l)(i) of this guideline;  average specific  growth  rate,  as described  in paragraph
       (f)(l)(ii) as described in this guideline; and area under the growth curve, as described in
       paragraph (f)(l)(iii) in this guideline.

              (i) Final population  density or yield. To correctly represent yield, the initial
              biomass values should be subtracted from the final biomass  values for  each test
              vessel. Since the initial values are extremely small relative to  the final  values, this
              correction  has a small  impact  upon the  test  results  but  is  nonetheless
              recommended.

                                        Y = bl -b0                              Equation 1

                    where:

                    7= final yield of observed biomass (cell density)

                    bo = biomass (cell density) at test initiation

                    b\ = biomass (cell density) at test termination

              (ii) Average specific growth rate.   Average specific growth rate (sometimes
              called relative growth rate) is the rate of growth over a given time interval.   The
              growth rate cell density for each test vessel (replicate) over a given time interval
              is calculated as given in Equation 2.  At a minimum, the average specific growth
              rate is calculated for the time interval between test initiation and termination.  The
              average  specific growth rate during the course of the test (days 0 to  1,  1  to 2,  2 to
              3, etc),  also called the section-by-section growth rate, is calculated also for each
              test vessel to  assess effects  on the pattern  of growth  of the test substance
              occurring during the exposure period, such as an increased lag phase.
                                        = __w,	^                         Equation 2
                                               t

                     where:

                     r;-;- = average specific growth rate per day (day"1) of observed biomass
                     (cell density) from time /' toy.

                     b{ = observed biomass (cell density) at beginning of the observation
                     interval, time /'



                                      Page 15 of 26

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              t>] = observed biomass (cell density) at end of the observation interval,
              timey

              t = time interval from / toy in days

       (iii) Area under the growth curve. The area under the growth curve for each
       test vessel (replicate) based on cell density is calculated using Equation 3.  At a
       minimum the area under the growth curve from test initiation through termination
       is calculated.


    A = (b^\tl)+(bl+b2~2A°\t2 -tl)+^1+b:~2b°\n -Ci)   Equation 3
              where:

              A = area under the growth curve for biomass (cell density)

              bo = observed biomass (cell density) at test initiation

              b\ = observed biomass (cell density) at time ti

              &2 = observed biomass (cell density) at time t2

              bn = observed biomass (cell density) at time tn

              t\ = time (units day) of the first measurement after test initiation

              U = time (day) of the nth measurement after test initiation

(2) Summary statistics—

       (i) Environmental conditions.  Calculate descriptive statistics (mean, standard
       deviation, coefficient of variation, minimum, maximum) by treatment level for
       temperature  and pH.  Calculate descriptive statistics (mean, standard deviation,
       coefficient of variation, minimum,  maximum) by  test vessel position for  light
       intensity.

       (ii) Test  substance  concentration.   Calculate  descriptive  statistics  (mean,
       standard  deviation, minimum, maximum, coefficient of variation) by test vessel
       and treatment level  of the  test substance soluble concentration.   For  each
       treatment level,  compare  the  initial  test  substance  concentration  with  test
       substance concentration at the end  of the test in each  treatment.  If the test
       substance was not stable calculate a rate of decline of the test substance; a time-
       weighted  mean concentration should be  calculated  under these circumstances
       (e.g.,  exponential decay calculate the  area  under  the  exponential decay
       concentration curve divided by the total exposure days).  For pesticides under
       unstable test substance conditions, the measured test substance concentration at
       test initiation is considered  appropriate rather than  the time-weighted average

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       because of the exposure estimate currently used  by OPP for calculating risk
       estimates.

       (iii) Cell density.  For each treatment level and observation time calculate the
       mean,  standard deviation,  and coefficient of variation  for algal cell  density.
       Calculate the mean treatment yield, average specific growth rate, and area under
       the growth curve based on changes in algal cell density from test initiation to test
       termination.  Calculate the mean treatment section-to-section growth rate for cell
       density.

       (iv) Appearance and condition. Morphological symptoms of plant injury should
       be summarized in tabular form by time of observation, treatment, and replicate.
       Definition of any index values used for morphological symptoms, indicating the
       severity of the symptom(s),  should be provided.

(3) Percent inhibition —

       (i) Treatment level.  For yield, average specific growth rate, and  area under the
       growth curve calculate the  percent inhibition (%I)  at  each treatment level at 96
       hours using Equation 4.
                             ..,                                       _    „    A
                             %/ = -i - ii — L                         Equation 4
              where:
              C = the control mean response value (yield, average specific growth rate,
              and area under the growth curve); and

              X = the treatment  mean response value (yield, average specific growth
              rate, and area under  the  growth curve, respectively).   Stimulation is
              reported as negative %I.

       (ii) Growth pattern.  If there are substantial differences between the section-by-
       section growth rates and the average growth rates for a test vessel  this indicates
       deviation from theoretical  exponential growth.  In this instance, compare specific
       growth rates from exposed cultures during the time period of maximum inhibition
       to those for controls during the same period.  The  same time interval should be
       used for each test vessel in all treatments.
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(4) Doubling time of controls. The doubling time (Td) of cell density in controls at test
termination is calculated using Equation 5.

                              Td = ln(2)/                               Equation 5
                                     / T 'Control

       where:

       r control  = mean of the control average specific growth rate test vessel values from
       paragraph (f)(l)(ii) of this guideline.

(5) Limit test—

       (i) ICso values. To ascertain that the yield, average specific growth rate, and area
       under the growth curve 96-hour  ICso values based on algal  cell  density occur
       above the "limit" concentration, a one-sided test which compares the difference
       between two sample  groups to a fixed value (or difference) is performed for each
       of these response measures.  For a  comparison of sample means, the difference
       defining the ICso compared to controls is operationally defined as a 50% reduction
       or  inhibition from the  control sample mean (Equation 6).   The null  hypothesis
       (Ho)  stated in  terms  of true population parameters is that  the difference of the
       "limit" treatment mean response (|iiimit) from the control mean response (^control) is
       greater than or equal  to a 50% reduction, compared to the control (i.e., HQ: |icontroi
       - Hiimit > So).  The alternative hypothesis (HA) is that this difference is less than a
       50% reduction, compared to the control (HA: Hcont-oi - Hiimit < So). An example of a
       parametric two-sample comparison test  is the  Student's  t-test.   If  the null
       hypothesis is rejected,  the inhibition level for the given response measure (i.e.
       yield or  the average specific growth  rate based  on  cell  density) in the limit
       treatment  as compared  to the control is declared to be less than 50% (i.e.., ICso >
       limit concentration).  If the null hypothesis is not rejected, the limit treatment as
       compared to the control response is declared to be 50% or greater (i.e.,  ICso <
       limit concentration).
                                                                        Equations

              where:

              So  = difference between two parameters, defined in this case as a p%
              reduction from the control sample mean;

              ^control = control sample mean response (e.g., yield or the average specific
              growth rate based on cell  density); and

              p = percent reduction from the control sample mean, which  is 50 in the
              caseofthelCso.
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       (ii) NOEC.  To ascertain that there is no observable effect at the limit treatment
       (i.e., NOEC > limit concentration) for a  given response measure (yield  or the
       average specific growth rate based on cell density), the limit treatment response is
       compared to the  control  treatment  response using  a one-sided two-sample
       parametric or nonparametric  test,  as  appropriate (see  OCSPP 850.4000).  The
       minimum significant difference detectable by the test or a similar estimate of the
       sensitivity of the test should be determined and reported.

       (iii) Multiple-dose definitive testing.

              (A) A multiple-dose definitive test  is performed for a given test species if
              either the effect or inhibition level for one or more response measures (i.e.,
              yield or the average specific growth rate based on cell density) in the limit
              treatment as  compared  to  the control  response at test termination are
              declared to  be 50%  or  greater effect  (i.e.,  the null  hypothesis  is not
              rejected) or the NOEC is less than the limit concentration.

              (B) Multiple-dose definitive testing may be waived for a given test species
              if at test termination the "limit" treatment response is both statistically less
              than a 50% decrease from the control response and there is no observable
              adverse effect from the control response for all measures of effect (yield or
              the average specific growth  rate based on cell density).

(6) Multiple-dose definitive test—

       (i) Concentration-response curve, slope  and ICso.  For concentration-response
       tests statistical  procedures are employed to  calculate the  96-hour ICso value
       (standard error and 95% confidence interval) for yield, average specific growth
       rate, and area under  the growth curve based on cell  density.  If a concentration-
       response curve model was fit to the data to determine an ICso value, the  model
       parameters  (e.g.,  slope)  and their uncertainty estimates (e.g.,  standard  error)
       should be recorded.  The values  for each  test vessel,  not the  mean for each
       treatment, should be used as the response variable in fitting the model.

       (ii) NOEC.  The 96-hour NOEC values for yield, average specific growth rate,
       and area under the  growth curve based  on cell density are  determined.  For
       pesticides if a 96-hour NOEC value can not be determined the 96-hour ICos value
       (standard error and 95% confidence interval) is estimated and used in place of the
       NOEC. For industrial chemicals, the specific ICX used in place of a NOEC that
       can not be determined will vary, consult with the Agency. Methods, assumptions,
       and results of the statistical approaches used should be recorded.

       (iii) Statistical methods.  Statistical procedures for modeling continuous toxicity
       data are available and should be used (see references in paragraphs (j)(2), (j)(4)
       and (j)(14) of this guideline and OCSPP 850.1000).   Additional discussion about
       endpoints and statistical  procedures is  found in OCSPP  850.1000 and OCSPP
       850.4000.
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              (iv) Algicidal and Algistatic concentrations.  If applicable, results from  the
              staining procedure  or the recovery phase are used to determine the algistatic
              concentration (refer to paragraph (e)(9)(v)(C) of this guideline).

(g) Tabular summary of test conditions.  Table 3 lists important conditions that should prevail
during the definitive test.  Except for the number of test concentrations,  Table 3 also lists  the
important conditions that should prevail during a limit test.  Meeting these test conditions will
greatly increase the likelihood that the completed test will be acceptable or valid.

       Table 3.—Summary of Test Conditions for the Algal Toxicity Test
Test type
Test duration
Test matrix
Temperature
Light quality
Light intensity
Photoperiod
Shaking
Salinity
Test vessel size
Test solution volume
Age of inoculum
Inoculum concentration
Number of replicate test vessels
per concentration
Test concentrations
Test concentration preparation
Measures of effect (Measurement
endpoints)
Static
96 hours
Synthetic growth medium appropriate for the test species
24 °C for P. subcapitata and N. pelliculosa; 20 °C for S. costatum.
Constant during test within ± 2 °C.
Cool-white fluorescent
60 umol/m2/s
Continuous light for P. subcapitata and N. pelliculosa; 14 hour
light:10 hour dark for S. costatum
Continuous at 100 oscillations per minute for P. subcapitata and N.
pelliculosa; manual, once or twice daily, for S. costatum
30 ± 5 ppt for saltwater species (S. costatum)
125 - 500 mL Erlenmeyer flasks
Less than or equal to 50% of the volume of the test vessel
From logarithmically-growing stock cultures (typically 3 - 7 days old)
10,000 cells per milliliter (cells/mL) for P. subcapitata and S.
costatum. At least 10,000 cells/mL for other species. Inoculum
volume less than 2 mL.
Four (minimum)
Unless performing limit test, minimum of 5 test concentrations plus
appropriate controls
Aqueous solutions prepared by adding test substance to synthetic
nutrient medium, directly or via vehicle
96-hour IC50 and NOEC (or IC05) values for yield, average specific
growth rate, and area under the growth curve based on algal cell
density
(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or
more of the conditions in Table 4 occurred or one or more performance objectives in Table 4
were not met. This list should not be misconstrued as limiting the reason(s) that a test could be
found unacceptable or invalid.  However, except for the  conditions listed in Table 4 and in
OCSPP 850.4000, it is unlikely a study will be rejected when there are slight variations from
guideline  environmental  conditions and study  design  unless  the  control  organisms  are
significantly affected, the precision of the test is reduced, the power of a test to detect differences
is  reduced,  and/or significant biases are introduced in defining the  magnitude of effect  on
measurement endpoints as compared to guideline conditions.   Before departing significantly
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from  this guideline, the investigator should  contact the Agency to discuss the reason for the
departure and the effect the  change(s)  will have on test acceptability.  In the test report, all
departures from the guideline should be identified, reasons for these  changes given, and any
resulting effects on test endpoints noted and discussed.

       Table 4.—Test Validity Elements for the Algal Toxicity Test
1. All test vessels and closures were not identical.

2. Treatments were not randomly assigned to test vessels, and test vessels were not randomly assigned
to positions in the growth chamber.

3.  A medium (untreated) control [and solvent (vehicle) control, when a solvent was used] was not
included in the test.

4.  The concentration of solvent in the range used affected growth of the test species.

5.  During the 96 hour test period, cell counts in the controls did not increase by a factor of at least 100
times for P. subcapitata and a factor of at least 30 times for S. costatum (i.e., logarithmic growth in the
controls was not reached during  the test).

6. A minimum of five test concentrations were not used in the definitive test.

7. Controls were contaminated with the test substance.

8. The lowest test concentration  level was not less than the 96-hour yield, average specific growth rate,
and area under the growth curve IC50 values based on cell density.

9.  For testing with industrial chemicals a surfactant or dispersant was used in the preparation of a stock
or test solution.

10. Temperature and light intensity were not measured as specified during the test.

(i) Reporting—

       (1) Background information.  Background  information to be supplied in the report
       consists at a minimum of those background information items listed in paragraph (j)0) of
       OCSPP 850.4000.

       (2) Guideline  deviations.  Provide a statement of the  guideline or protocol followed.
       Include a description of any  deviations from the test guideline or any occurrences which
       may have influenced  the results of the  test, the reasons  for these changes, and any
       resulting effects on test endpoints noted and discussed.

       (3) Test substance.

              (i) Identification of the test substance:  common  name, IUPAC  and CAS names,
              CAS number,  structural formula, source, lot or batch number, chemical state or
              form  of the test substance,  and  its purity  (i.e.  for pesticides,  the identity and
              concentration of active ingredient(s)).
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       (ii) Storage conditions of the test chemical or test substance and stability of the
       test chemical or test substance under storage conditions if stored prior to use.

       (iii)  Methods  of  preparation  of the test  substance,  stock solutions, and  the
       treatment concentrations used in the range-finding and definitive test, or limit test.

       (iv) If a vehicle (solvent) is  used to prepare stock or test  substance provide:  the
       name and source of the vehicle, the nominal concentration(s) of the test substance
       in the vehicle in stock solutions or mixtures, and the vehicle concentration(s) used
       in the treatments and solvent  control.

(4) Plant test species.

       (i) Scientific and common name, plant family, and strain.

       (ii) Source and  method of species and strain verification.

       (iii) Culture practices, including culturing media used, and conditions.

       (iv) Acclimation period, if applicable.

       (v) Age (stage) of inoculum at test initiation.

(5) Test system and conditions.  Description of the test system and conditions used in
the definitive or limit test, and any preliminary range-finding tests.

       (i) Description  of the growth chamber,  or laboratory location, type of lights, and
       oscillation rates and type of apparatus.

       (ii) Description of the test vessels: size, type, material, fill volume.

       (iii) Volume of test solution in the test vessels.

       (iv) Description of preparation of inoculum used to begin test.

       (v) Inoculum volume and density added to each test vessel.

       (vi) Number of test vessels (replicates) per treatment level and control(s).

       (vii) Description of the preparation of the synthetic growth media used including
       the preparation date, concentration of all constituents, the  initial pH, and storage
       conditions and duration prior to use in test.

       (viii) Description of the dilution water and any water pretreatment: source/type;
       pH;  total  organic carbon  content; particulate matter content;  metals, pesticides,
       and  chlorine concentration.    Describe the frequency and  sample date(s)  for
       documenting dilution water quality.
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       (ix) Methods used for treatment randomization and assignment of inoculum to test
       vessels.

       (x) Date of introduction of test organisms to test solutions and test duration.

       (xi) Exposure technique.

       (xii) The photoperiod and light source.

       (xiii) Methods and frequency of environmental  monitoring performed during the
       definitive or limit study for temperature, light intensity, pH.

       (xiv) Methods and  frequency of measuring test  substance to confirm exposure
       concentrations.

       (xv) Methods  and frequency  of  measuring  cell density,  and  any  other
       measurements or observations of phytotoxic symptoms.

       (xvi) For the definitive and  limit  test,  all  analytical procedures  should be
       described.  The  accuracy of the method, method detection limit,  and limit of
       quantification should be given.
(6) Results.
       (i) Tabulation of test substance analytical results by test vessel and treatment
       (provide raw data) and descriptive statistics (mean, standard deviation, minimum,
       maximum, coefficient of variation).

       (ii) Environmental monitoring data results (test solution or air temperature, light
       intensity, and pH) in tabular form (provide raw data for measurements not made
       on a  continuous basis), and descriptive  statistics (mean, standard  deviation,
       minimum, maximum).

       (iii) For preliminary range-finding tests, if conducted,  the cell density  at each
       treatment level  and in the control(s).  A description and count of morphological
       phytotoxic effects, if recorded, at each treatment level and in the control(s).

       (iv) For a limit test, tabulate for the limit concentration and  the control(s) by
       replicate, the cell density at test initiation, at 24,  48, and 72 hours,  and at test
       termination, the treatment means and  standard deviations (provide the raw data).

       (v) For the definitive test, tabulation by test vessel and treatment of cell density at
       test initiation, at 24, 48, and 72 hours, and at test termination and treatment means
       and standard deviations (provide the raw data).

       (vi) For the limit and definitive tests, tabulation by test vessel and treatment of
       yield, average specific  growth rate,  and area under the  growth curve  for cell
       density.
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(vii) For the limit and definitive  tests, tabulation  of the mean treatment yield,
average specific growth rate, and  area under the growth curve values, treatment
standard deviations for these variables, and the treatment %I (or stimulation) in
yield, average specific growth rate, and area under the growth curve relative to the
control values.

(viii) For the limit and definitive test, tabulation of observed morphologic signs of
toxicity (size, abnormal shape or color changes, and any other observed effect) by
observation time, treatment level and replicate.

(ix) Graphs of the 96-hour concentration-response data for yield, average specific
growth rate, and area under the growth curve based on cell density.

(x) For a limit test, provide the results of hypothesis tests.

(xi)  For the limit test, provide a description  of the  statistical  methods  used
including software package, and the basis for the choice of method.

(xii) For the definitive study and for those effect measures (yield, average specific
growth rate, and area  under the growth curve for cell density) with data sufficient
to fit  a concentration-response  relationship,  tabulation  of the slope  of the
concentration-response  curve and its standard  error and 95% confidence limits
and any goodness of fit results.

(xiii) For the definitive test, tabulation  of 96-hour ICso values for yield, average
specific growth rate, and area under the growth curve for cell density.

(xiv) For the definitive test, a tabulation of the  96-hour NOEC and LOEC values
for each response variable (yield, average  specific growth rate, and area under the
growth curve based on cell density).  For pesticides, the ICos  and 95% confidence
interval should  be  reported for response data where  an  NOEC could  not be
determined.

(xv) Description of  statistical  method(s) used for  point estimates, including
software package, for determining ICso values, fitting the  dose-response model,
and the basis for the choice of method.  Provide results of any goodness-of-fit
tests.

(xvi) Description  of  statistical  method(s)   used  for  NOEC   and  LOEC
determination, including  software package, and the basis  for the  choice of
method.  If an ICos value is used in  place of a NOEC provide a description of
statistical method(s)  used for point estimates,  including software package, for
determining ICos values, fitting the  dose-response  model,  and the basis  for the
choice of method. Provide results of any goodness-of-fit tests.

(xvii) If determined, report the algistatic and algicidal concentrations.
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(j) References.   The following references  should be consulted for additional background
material on this test guideline.

       (1) American Society for Testing and  Materials.  ASTM E 1218-04. Standard guide for
       conducting 96-h toxicity tests with microalgae.  In:  Annual Book of ASTM Standards,
       Vol. 11.06, West Conshohocken, PA. Current edition approved 2004.

       (2) Bruce, R.D. and DJ. Versteeg, 1992. A statistical procedure for modeling continuous
       toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.

       (3) Miller, W.E., J.C. Greene and T. Shiroyama, 1978. The Selenastrum capricornutum
       Printz  Algal  Assay  Bottle  Test:   Experimental  Design,  Application,  and  Data
       Interpretation Protocol. EPA-600/9-78-018. Corvallis, OR.

       (4) Nyholm, N.,  P.S.  Sorenson,  K.O. Kusk, and E.R.  Christensen, 1992.   Statistical
       treatment of data from microbial toxicity tests. Environmental Toxicology and Chemistry
       11:157-167.

       (5) Organization for Economic Co-operation and Development, 2006. OECD Guidelines
       for  Testing  of Chemicals,  TG  201, Freshwater  Alga and  Cyanobacteria,  Growth
       Inhibition Test. Updated guideline, adopted 23 March, 2006.

       (6) Payne, A.G. and R.H. Hall,  1979.  A method for measuring  algal toxicity and its
       application to the safety assessment of new chemicals,  pp 171-180 in L.L. Marking and
       R.A. Kimerle (eds.).  Aquatic Toxicology,  ASTM STM 667, American Society  for
       Testing and Materials, Philadelphia, PA.

       (7) Provasoli, L., 1963.  Growing marine seaweed, Proceedings of the 4th International
       Seaweed Symposium, Pergamon Press, Vol. 4, pp. 9-17.

       (8) Stein, J., Handbook of Phycological Methods, Cambridge University Press.

       (9) U.S. Environmental Protection Agency, 1974. Marine Algal Assay Procedure: Bottle
       Test, Eutrophication  and  Lake Restoration  Branch, Pacific Northwest Environmental
       Research Center, Corvallis, OR.

       (10) U.S. Environmental Protection Agency,  1978.  Bioassay Procedures for the Ocean
       Disposal Permit Program, EPA-600/9-78-010, Environmental Research Laboratory, Gulf
       Breeze, FL.

       (11) U.S. Environmental Protection Agency, 1982.  Pesticide Assessment Guidelines
       Subdivision J Hazard Evaluation: Nontarget Plants.  Office of Pesticides and  Toxic
       Substances, Washington, D.C.  EPA-540/9-82-020, October 1982.

       (12) U.S. Environmental Protection Agency, 1986. Hazard Evaluation Division Standard
       Evaluation Procedure, Non-target Plants: Growth and Reproduction of Aquatic Plants -
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Tiers 1  and 2.  Office of Pesticides Programs,  Washington, D.C.  EPA 540/9-86-134,
June 1986.

(13) U.S.  Environmental Protection Agency, 1994.  Pesticides Reregi strati on Rejection
Rate Analysis: Ecological Effects, Office of Prevention, Pesticides and Toxic Substances,
Washington, D.C. EPA 738-R-94-035

(14) VanEwijk, P.H. and J.A. Hoekstra, 1993. Calculation of the EC50 and its confidence
interval when subtoxic stimulus  is present. Ecotoxicology and Environmental Safety
25:25-32.

(15) Walsh, G.E. and S.V. Alexander, 1980.  A marine algal bioassay method: results
with pesticides and industrial wastes. Water, Air and Soil Pollution 13:45-55.
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