August, 1982
          WASHINGTON,  D.C.  20460

Office of Toxic Substances                                  EG-8
Guideline for Testing Chemicals                     August, 1982

                    ALGAL ACUTE  TOXICITY TEST
    (a)  Purpose.  The  guideline in this 'section'is intended for
use in developing data  on  the  acute toxicity of  chemical
substances and mixtur'es  ("chemicals")  subject to environmental
effects test regulations under the  Toxic Substances Control Act
(TSCA) (P.L. 94-469,  90  Stat.  2003, 15 U.S.C. 2601 et seq.).
This guideline prescribes  test procedures and conditions using
freshwater and marine algae  to develop data on the phy to toxicity"
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of chemicals.  The United  States Environmental Protection Agency
(USEPA) will use data from these tests in assessing the hazard of
a chemical to the environment.
    (b).  Def initions .   The definitions in Section 3 of the Toxic
Substances Control Act  (TSCA)  and  the  definitions  in Part 792—
Good Laboratory' Practice Standards  apply to this test
guideline.  The following definitions  also  apply to this
    (1)   "Algicidal" means  having  the  property of  killing algae.
    (2)   "Algistatic" means having  the property of  inhibiting
algal growth.
    (3)   "ECx" means the experimentally derived chemical
concentration that is calculated to effect  X percent of the test

                                                    August, 1982

    (4)  "Growth" means  a  relative  measure of  the viability of an

algal population based on  the  number  and/or weight of  algal cells

per volume of nutrient medium  or  test solution in a specified

period of time.

    (5)  "Static system" means  a  test contai er in which the test

solution is not renewed  during  the  period  oL the  test.

   . (.c)  Test procedures — (1)  -Summary of  the  test.  (A)  In

preparation for the test,  fill  test containers  with appropriate

volumes of nutrient medium and/or test solution.   Start the test

by introducing algae into  the  test  and control  containers  in the

growth chambers.  Environmental conditions within the  growth

chambers are established at predetermined  limits.

    (3)  At the end of 96  hours enumerate  the  algal cells  in all

containers to determine  inhibition  or stimulation of growth in

t'.st containers compared to controls.   Use data to define  the

concentration-response curve,  and calculate the EC-10, EC-50, and

EC-90 values.

    (2)   [Reserved]

    (3)  Range-finding test.   (i)   A  range-finding test should be1

conducted to determine if:

    (A)  definitive testing is  necessary

    (B)  test chemical concentrations  for  the  definitive test.

                                                    August,  1982
    (ii)  Algae are exposed  to a widely spaced  (e.g.,  log

interval) chemical concentration series.   The  lowest value  in the

series, exclusive of controls, should be  at  the  chemical's

detection limit.  The upper  value,  for water soluble compounds,

should be the saturation concentration.   No  replicates  are

required; and nominal concentrations of the  chemical are

acceptable unless definitive testing is not  required.

    (iii)  The test is performed once for each  of  the  recommended

algal species or selected alternates.  Test  chambers should
                         I                                 •   i
contain equal volumes of test solution and 'approximately 1  x 104

Selenastrum cells/ml or 7.7  x 104  Skeletonema cells/ml  of test

solution.  The algae should  be exposed to each  concentration of

test chemical for up to 96 hours.   The exposure  period  may be

shortened if data suitable for the  purposes  of  the range-finding

test can be obtained in less time.

    (iv)  Definitive testing is not necessary  if the highest

chemical concentration tested (water saturation  concentration or

1000 mg/1) results in less than a  50 percent reduction in growth

or if the lowest concentration tested (analytical  detection

limit) results in greater than a 50 percent  reduction  in growth.

    (4)  Definitive test.  (i)  The purpose  of  the definitive

test is to determine the concentration response  curves,  the  EC-

10's, EC-50's, and Ec-90's for algal growth  for  each species

                                                  .  August,  1982
tested, with a minimum amount of testing beyond  the  range-finding


    (ii)  Algae should be exposed  to  five or  more  concentrations

of the test chemical in a geometric series  in  which  the  ratio  is

between 1.5 and 2.0 (e.g., 2, 4, 6, 8, 16,  32  and  64 mg/1).

Algae should be placed in a minimum of three replicate test

containers for each concentration  of  test chemical and control.

More than three replicates may be  required  to  provide sufficient

quantities of test solution for .determination  of test substance

concentration at the end of the test. ''Each test chamber should

contai-n equal volumes of test solution and  approximately 1 x  104

Selenastrum cells ml"1 or 7.7 x 104 Skeletonema  cells/ml of test

solution.  The chemical concentrations should  result in  greater

than 90 percent of algal growth being inhibited  or stimulated  at

the lowest concentrations of test  substance compared to  controls.

    (iii)  Every test should include  a control consisting of  the

same nutrient medium, conditions,  procedures,  and  algae  from  the

same culture, except that none of  the test substance is  added.

If a carrier is present in a-ny of  the test  chambers,  a separate

carrier control is required.

    (iv)  The test begins when algae  from seven  to ten-day-old

stock cultures are placed in the test chambers containing test

solutions having the appropriate concentrations  of the test

                                                    August,  1982
substance.  Algal growth in controls should  reach  the  logarithmic

growth phase by 96 hours (at which  time  the  number of  algal  cells

should be approximately 1.5 x 106/ml for  Skeletonema or 3.5  x

106/ml for Selenastrum).   If growth in controls  does not reach

this logarithmic phase within this  96-hour period,  the test  is

invalidated and should be  repeated.  At  the  end  of  96  hours  the

algal growth response (number or  weight  of algal cells/ml)  in all

test containers and controls should be determined  by an indirect

(spectrophotometry, electronic cell  counters, dry  weight, etc.)
or a direct (actual microscopic cell count)  method.  Indirect

methods should be calibrated by a direct  microscopic count.   The

percentage inhibition or stimulation of  growth  for each

concentration, EC-10, EC-50, EC-90  and the concentration-response

curves are determined from these  counts.

    (v)  At the end of the definitive test,  the  following

additional analyses of algal growth response should be performed:

    (1)  Determine whether the altered growth response between

controls and test algae was due to  a change  in  relative cell

numbers, cell sizes or both.  Also  note any  unusual cell shapes,

color differences, f locculations, adherence  of  algae to test

containers, or aggregation of algal  cells.

    (2)  In test concentrations where growth is maximally

inhibited, algistatic effects may be differentiated from


                                                    August,  1982 .
algicidal effects by the following  two methods:

    (A)  Add 0.5 ml of a 0.1 percent solution  (weight/volume)  of

Evans blue stain to a one milliliter aliquot of  algae  from a

control container and to a one milliliter  aliquot  of  algae from

the test container having the lowest concentration of  test

chemical which completely inhibited algal  growth (if  algal growth

was not completely inhibited, select an aliquot  of  algae  for

staining from the test container  having the highest concentration

of test chemical which inhibited  algal growth).  Wait  ten to

          '••        '          i             •          i.
thirty minutes, examine microscopically, and determine the

percent of the cells-which stain  blue (indicating  cell

mortality).  A staining control should be  performed concurrently

using heat-killed or formaldehyde-preserved algal  cells;  100

percent of these cells should stain blue.

    (B)  Remove 0.5 ml aliquots of test solution containing

g rowt h-i nh ib i ted algae from each  replicate test  container having

the concentration of test substance evaluated  in (2) (I) above.

Combine these aliquots into a new test container and  add  a

sufficient volume of fresh nutrient medium-to  dilute  the  test

chemical to a concentration which does not affect  growth.

Incubate this subculture under the environmental conditions used

in the definitive test for a period of up  to nine  days, and

observe for algal growth to determine if the algistatic effect

                                                    August, 1982
noted after the 96-hour test  is  reversible.   This  subculture test

may be discontinued as soon as growth  occurs.

    (5)  [Reserved]

    (6)  Analytical measurements — (i)   Chemical.   (A)   Glass

distilled or deionized water  should  be  used  in  the preparation of

the nutrient medium.  The pH  of  the  test  solution  should be

measured in the control and test containers  at  the beginning and

at the end. of the definitive  test1.   The concentration  of test

chemical in-the test containers should  be  determined at the
 I  i .     i   j |                                           I
beginning and end of the definitive  test  by  standard analytical

methods which have been validated prior to the  test.   An

analytical method is unacceptable  if likely  degradation products

of the chemical, such as hydrolysis  and oxidation  products, give

positive or negative interference.

    (B)  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 concentration of test chemical determined.   In addition, the

concentration of test chemical associated  with  the algae alone

should be determined.  Separate and  concentrate  the algal cells

from the test solution by centrifuging  or  filtering the remaining

                                                    August,  1982
pooled sample and measure the test substance concentration  in  the

algal-cell concentrate.

    (ii)  Numerical.  Algal growth response  (as  percent of

inhibition or stimulation in the test solutions  compared to the

controls) is calculated at the end of the  test.   Mean  and

standard deviation should be calculated and  plotted  for each

treatment and control.  Appropriate statistical  analyses should

provide a goodness-of-fit determination for  the  concentration

response curves.  The concentration response curves  are plotted

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using the mean measured test solution concentrations obtained  at

the end of the test.         .      -

    (d)  Test conditions—(1)  Test species .   Species  of algae

recommended as test organisms for this test  are  the  freshwater

green alga, Selenastrum capr icornu turn, and  the marine  diatom,

Skeletonema costatum.  Algae to be used in acute  toxicity tests

may be  initially obtained from commercial  sources  and

subsequently cultured using sterile technique.   Toxicity testing

should  not be performed until algal cultures are shown to be

actively growing (i.e. capable of logarithmic  growth within the

test period) in at  least two subcultures lasting  seven days each

prior to the start  of the definitive test.   All  algae  used  for a

particular test should be from the same source and  the same stock

culture.  Test algae should not have been used in  a  previous



                                                    August, 1982
test, either in a treatment  or  a  control.

     (2)  Facilities — (i)  General.   (A)   Facilities needed to

perform this test includes:   a  growth  chamber or a controlled

environment room that  can hold  the  test containers and will

maintain the air temperature, lighting intensity and photoperiod

specified in this test guideline; apparatus for culturing and

enumerating algae; a source  of  distilled and/or deionized water;

and  apparatus for carrying out  analyses  of  the test chemical.

     (B)  Disposal facilities  should  be adequate to accommodate

   '  :           !   i     |  i
spent glassware, algae and' test solutions  at the end of the test

and  any bench covering,  lab  clothing,  or other contaminated

ma terials.

     (ii)  Test containers.   Erlenmeyer flasks should be used for

test containers.  The  flasks  may  be  of any  volume between 125 and

500  ml as long as the  same size is  used  throughout a test and the

test solution volume does not exceed 50  percent of the flask

volume .

     (iii)  Cleaning and  sterilization.  New test containers may

contain substances which inhibit  growth  of  algae.  They should

therefore be cleaned  thoroughly and  used several times to culture

algae before being used  in toxicity  testing.   All glassware used

in algal culturing or  testing should be  cleaned and sterilized

prior to use'according  to standard good  laboratory practices.



                                                   August,  1982
    (iv)  Conditioning.  Test containers should be conditioned  by

a rinse with the appropriate test solutions prior to the start  of

the test.  Decant and add fresh test solutions after an appro-

priate conditioning period for the test chemical.

    (v)   Nutrient medium.  (A)  Formulation and sterilization  of

nutrient medium used for algal culture and preparation of test

solutions should conform to those currently recommended by the

U.S. EPA for freshwater and marine algal bioassays.  No chelating

agents should be included in the nutrient medium used for test

 I i  !       I                -             I ,<                  |
solution preparation.  Nutrient medium should be freshly prepared

for algal testing, and may be dispensed in appropriate volumes  in

test containers and sterilized by autoclaving or filtration.   The

pH of  the nutrient medium should be 7.5 for Selenastrum and 8.1

for Skeletonema at the start of the test and may be adjusted

prior  to test chemical addition with 0.1N NaOH or HC1.

    (B)  Dilution water used for preparation of nutrient medium

and test solutions should be filtered, deionized or glass

distilled.  Saltwater for marine algal nutrient medium and test

solutions should be prepared by adding a commercial, synthetic,

sea salt formulation or a modified synthetic seawater formulation

to distilled/deionized water to a concentration of 30 parts per


    (vi)  Carriers .  Nutrient medium should be used in making



                                                    August,  1982
stock solutions of the test  chemical.   If  a  carrier  other  than

nutrient medium is absolutely necessary to dissolve  the  chemical,

the volume used should not exceed  the  minimum  volume necessary to

dissolve or suspend the chemical  in  the test solution.

    (3)  Test parameters.  (A)  The  test temperature should  be

maintained at 24°±l°c for Selenastrum  and  20°±1°C  for

Skeletonema.  Temperature should be  recorded hourly  during the

test.                  '                 •

    (B)  Test chambers containing  Selenastrum  should be
illuminated continuously and those containing  Skeletonema should

be provided a 14-hour light and 10-hour dark photoperipd with a

              .'  i
30 minute transition period under fluorescent  lamps  providing 300

±25 uEin/m^ sec (approximately 400  ft-c)  measured  adjacent to

the test chambers at the level of test solution.
          .  t

    (C)  Stock algal cultures should be shaken twice daily  by

hand.   Test containers should be placed on a rotary  shaking

apparatus and oscillated at approximately  100  cycles/min for

Selenastrum and at approximately 60  cycles/min for  Skeletonema

during the test.  The rate of oscillation  should  be  determined at

least once daily during testing.

    (D)  The pH of nutrient medium in which algae are subcultured

should be 7.5 for Selenastrum and 8.1 for  Skeletonema, and  is  not

adjusted after the addition' of the algae.  The pH of  all test


                                                    August,  1982
solutions and controls should be measured at  the  beginning  and

end of the test.

    (E)'  Light  intensity should be monitored  at  least daily

during the test at the level of the test solution.

    (e)  Reporting .  The sponsor should submit  to the EPA all

data developed  by the test that are suggestive or predictive  of

acute phytotoxicity.  In addition to  the general  reporting

requirements prescribed in Part 792—Good Laboratory  Practice

Standards , the  following should be reported:
    (i) Detailed 'information about the test organisms, 'including'

the scientific name, method of verification,  and  source; .  -

    (ii)  A description of the test  chambers  and  containers  ,  the

volumes of solution  in the containers, the way  the  test  was  begun

(e.g. conditioning,  test substance additions, etc.),  the  number

of replicates, the temperature,  the  lighting, and method  of

incubation, oscillation rates, and type of apparatus;

    (iii)  The concentration of  the  test  chemical in  the  control

and in each treatment at the end of  the test  and  the  pH  of  the


    ( iv)  The number of algal cells  in.each treatment and  control

and the method used  to derive these  values at the beginning  and

end of the test; the percentage  of inhibition or  stimulation of


                                                    August/  1982
growth, relative to controls; and other  adverse  effect .in  the

control and in each treatment;

    (v)   The 96-hour EC-10,  EC-50  and EO90  values  and -their  95-

percent confidence limits, the methods  used  to  derive these

values,  the data used to define  the  shape  of  the  concentration-

response curve and the goodness-of-fit  determination;

    (vi)  Methods and data records of all  chemical  analyses of

water quality and test substance concentrations,  including method

validations and reagent blanks;

                     '. I       ! !   :    :      i              i
    (vii)  The results of any optional  analyses such as:  '

microscopic appearance of algae, size or color  changes, percent

mortality of cells and the fate  of subcultured  cells, the

concentration of test substance  associated with algae and test

solution supernate or filtrate;

    (viii)  If the range-finding test showed  that the highest

concentration of the chemical tested (not  less  than 1000  mg/1 or

saturation concentration) had no effect on the  algae, report  the

results  and concentration and a  statement  that  the  chemical is of

minimum phytotoxic concern;

    (ix)  If the range-finding test  showed greater  than a 50

percent inhibition of algal  growth at a test  concentration below

the analytical detection limit,  report  the results,

concentration, and a statement that  the chemical  is phytotoxic

below the analytical detection limit.