ES-5
                                   August, 1982
        TECHNICAL  SUPPORT DOCUMENT

                    FOR

         ALGAL, ACUTE TOXICITY  TEST
        OFFICE OF TOXIC  SUBSTANCES
OFFICE OF  PESTICIDES AND TOXIC  SUBSTANCES
   U.S.  ENVIRONMENTAL  PROTECTION SGENCY
          WASHINGTON,  D.C.  20460

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                                   ES-5
                                   August, 1982
        TECHNICAL  SUPPORT DOCUMENT

                    FOR

        ALGAL ACUTE TOXICITY  TEST
     _  OFFICE OF TOXIC  SUBSTANCES
OFFICE OF  PESTICIDES AND TOXIC  SUBSTANCES
   U.S.  ENVIRONMENTAL  PROTECTION AGENCY
          WASHINGTON,  D.C.  20460

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                      TABLE OF CONTENTS

         Contents                                     Page
I.      Purpose                                          1
II.     Scientific Aspects                               2
       Test Procedures                                  3
       General                                          3
       Range-finding Test                               6
       Definitive Test                                  7
       Analytical Measurements                          9
       Test Conditions                                  10
       Test Species                                     10
       Facilitites                                      14
       Test Containers                            I      15
       Cleaning and Sterilization                       15
       Conditioning                                     16
       Nutrient Medium                                  16
       Environmental Conditions                         17
       Reporting                                        20
III.   Economic Aspects                                 20
IV.     References                                       22

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Office of Toxic Substances                              ES-5
                                                August,  1982
  TECHNICAL SUPPORT DOCUMENT  FOR ALGAL ACUTE TOXICITY TEST
    Purpose
    The purpose of  this  document is  to provide the
scientific background and  rationale  used  in the development
of Test Guideline EG-8 which  uses  freshwater and marine
algae to evaluate the acute toxicity of  chemical
substances.  The Document  provides an account of the
scientific evidence and  an explanation of  the logic used in
the selection of the test  methodology, procedures and
conditions prescribed in the  Test Guideline.   Technical
                              i
issues and practical cons id er'at ions  relevant to the Test
Guideline are discussed.   In  addition,  estimates of the cost
of conducting the tests  are provided.
II. Scientific Aspects
    A.  Test Procedures
         1.  General.  A balanced  growth of  algae in the
aquatic environment  is essential,  but  extremes  in
productivity may be  detrimental  to  other organisms.   Some
algae are able to inhibit or stimulate  the growth of other
algae, for example Selenastrum can inhibit iMicrocys tis
growth in eutrophic  water (Toerien  et  al.  1974).   Inhibition
of algal growth would alter the  food web and  reduce  the
productivity of ecosystems.  The toxic  effect of  a chemical
or other inhibitor may increase  the susceptibility of algae
to other environmental stresses  (Fisher  and Wurster  1973).
Stimulation of algal growth may  cause  an algal  bloom which
may have negative aesthetic effects; may adversely affect
commercial sport fisheries (Lightner 1978, Lovell 1979)  and
recreation; may impart unpleasant  taste  to drinking  water;
may release substances deleterious  to  aquatic animals,

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 and/or  may  indirectly kill aquatic organisms by creating
 anoxic  conditions  (Shilo 1964,  Schwiramer and Schwimmer
 1967).   Stimulation of algal growth,  while primarily a
 problem in  eutrophic freshwaters, has created serious
 ecological  problems in the open ocean as well.  In the
 spring  of J.976 and extending into the fall, there was an
 extensive algal bloom, dominated by Ceratium tripos, located
 off  the New Jersey coast.  The bloom, together with a dearth
 of storm activity, anomalous surface wind conditions, and
 unusually warm sea surface temperatures resulted in a huge
Anoxic  area, 100 miles long and 40 miles |Wide which had a
 severe  impact on the finfish and shellfisn populations  in
 the  area.   The immediate effects on commercial and sport
 fishes, lobsters,  and shellfish were not entirely known.
 However, an estimated 59,000 metric tons of surf clams  were
 killed  (representing twice the annual U.S. harvest), and up
 to  50%  of other shellfish populations sampled were Killed.
 One  commercial trawler reported up to 75% of fish collected
 were dead.   It was predicted that these mortalities wou1d
 affect  recruitment, population size and harvests for years
 to come (Sharp 1976).
     Another more commonly known phenomenon is the adverse
 effect  caused by stimulated growth of toxigenic marine
 algae.   Frequently explosive mass development of these
 organisms  in the form of blooms and tides occur, resulting
 in fish kills, contaminated shellfish, and outbreaks of
 paralytic shellfish poisonings in humans.  (Shilo 1964,
 Taylor and  Seliger 1979).
     Even when toxigenic organisms are not present in
 sufficient  concentrations to affect human health, red tides

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may reduce the market for shellfish because of  adverse
publicity (Council on Environmental Quality,  1979).
Furthermore, the high concentrations of phytoplankton that
occur during blooms can be harmful  to shellfish because  the
rate of water transport by molluscs is reduced  and feeding
ceases (Galtsoff 1964).
    Algal growth was selected to measure phytotoxicity for
the following reasons:
    o    The selection of phytoplanktonic  algae for  toxicity
         testing is based upon their importance  in aquatic
         ecosystems.  Algae were one of the first cellular
         life forms, dating as far back as 3.1  billion years
         in the fossil record (Bold and Wynne 1978)  and  are
         numerous today.  Because phytoplankton  are
         ubiquitous, it is usually the case that most marine
         and freshwater ecosystems are based upon the
         primary production of phytoplankton  (Stern  and
         Stickle 1978).  Primary production is  of prime
         significance to estuarine energetics since  the
         primary producers are at the base of the food
         web.  In estuaries phytoplankton  are the main
         primary producers in the water (Vernberg 1977).
         Algae convert inorganic carbon to organic carbon
         and liberate oxygen during photosynthesis.  Thus,
         tney are primary producers of food and  energy for
         the lower trophic-level herbivores which in turn
         provide food for the upper trophic-level
         carnivores, generally fishes (Vance and Maki
         1976).  Some species fix nitrogen, required for the
         growth of vascular plants.  Therefore, much of the

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food people eat and the oxygen they breathe are  the
result of algal productivity.
Inferences may be drawn from laboratory tests  for
inhibition or stimulation of algal growth  as  to  the
extent to which a chemical substance can interfere
with primary productivity and nutrient cycling  in
lakes, streams, estuaries, and oceans.  Further
inferences may be drawn from algal bioconcentration
data as to the potential of a chemical substance to
bioaccumulate  in food chains.  However, in the
natural environment there are too many factorsj
acting to regulate algal populations which cannot
be simulated in a simple laboratory test.   The  real
value of  the test guideline  is to determine
thresnold toxicity values and to evaluate  the
relative  toxicity of test substances to one another
under rigidly controlled conditions.
Algal testing has been well  established in the
literature.  In 1967, the EPA began developing
algal assays for evaluating  the ecological effects
of pollution to the environment.  Initially
designed  for considering problems associated  with
eutrophication (Maloney and  Miller 1975),  algal
assays have also been used  to define the toxic
effects of heavy metals (Davies 1978), pesticides
(Schauberger and Wildman 1977, Walsh and Alexander
1980), oil spills  (Corner 1978, Fisher and Wurster
1973, O'Brien  and Dixon 1976, Vandermeulen and
Ahern 1976), chemical substances (US EPA 1978  a,b,c,
Harding and Phillips 1978), dyes (Little and

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         Chillingworth  1976),  complex  industrial wastes
         (USEPA 1978d,  Walsh  and  Alexander 1980, Walsh et
         al.  1980} and  natural organic components of  fresh
         and  marine  water  (Prakash and Rashid 1968).   Over
         the  years,  extensive  use of this  test has
         sufficiently refined  it  to qualify as a standard
         method to measure  water  quality.   Algal assays are
         recommended for use  by the APHA (1975) USEPA (1977,
         1978 a,b,c,d)  and  are currently under review by the
         American Society  for  Testing  and  Materials.
         Further discussion on thei validity of , applying      |
         algal assays in water quality assessment is  found
         in Fitzgerald  (1975); Joint Indus try/Government
         Task Force  on  Eutrophication  (1969); Leischman et
         al (1979);  USEPA  (1978b)  Miller et al. (1978);
         Murray et al.  (1971); Reynolds  et al. (1974);  and
         USEPA (1971, 1975a).
    o    The  algal growth method  is 1)  relatively rapid,  2)
         inexpensive, 3) capable  of being  performed by
         persons with minimal  technical  training and  4)
         reproducible,  using large  numbers  of organisms with
         sufficient  replication and precision.
    The test procedure  involves assessment  of algal growth
in test chambers relative  to controls  by requiring a
quantitative determination  of  algal cell numbers,  and  by
recommending  a) a qualitative  appraisal  of  algal numbers  and
size by means of microscopic observation,  and b) a
determination of viability  of  growth-inhibited algae  by
means of mortal staining coupled  with  microscopic
observation and/or subcultur ing .   The  test  procedure  is

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simple because it requires only the combination  of  set
amounts of test substance, nutrient medium  and  algae,  and
then monitoring the growth response 96 hours  later.   At the
end of 96 hours a further  assessment  of  growth  and  viability
is recommended.
    In the test the following  procedures are  required:
    o    Algal growth should be logarithmic at  the  beginning
         of  the test and algal number should  be determined.
    o    The  number of  algae should be determin-ed at the end
         of  the test.
    jo    The  concentration of  chemical  in, the test  solutiojn  \
         should be determined  at  the  beginning  and  end of
         the  test and  the  concentration  of  chemical
         associated with the algal cells should also be
         determined .
    o    growth and bioconcentration  data should be
         subjected  to statistical analyses.
    These  requirements  will  ensure  consistency  and  will
minimize variabili y of the  test  results.   The  test also
recommends testing of  algicidal  and/or algistatic chemical
effects.
         2.   Range-Find ing Test
    It  is  recommended  that a range-finding  test be  conducted
prior  to the  definitive test in  those instances where no
information  is  available or  can be  elucidated on the
phototoxicity of  the test  chemical.   This  approach  should
minimize  the possibility that  an  inappropriate  concentration
series will  be utilized in the definitive test  and  under
certain  circumstances  may  even preclude  the need to conduct
the definitive  test.   In order to minimize  the  cost and time

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required to obtain the requisite  data nominal  concentrations
are permitted, test duration may  be  shortened,  replicates
are not required and other test procedures  and  conditions
are relaxed.
    If test results indicate that the chemical  is  non-toxic
or very toxic to algae and if definitive  testing is  not
conducted, it is necessary to ascertain  that  the control
algae have attained a logarithmic  growth  rate  by 96  hours
and that the test was conducted at the specified incubation
temperature.  These verifications  establish that the algae
tes ted were viable and that the test was  properly  conducted.
    In some situations there may  be  enough  inf or nation
available on toxicity to select the  appropriate concen-
tration without a range-finding test.  The  range-finding
test (or other available information) needs to  be  accurate
enough to ensure that dose levels  in the  definitive  test are
spaced to result in concentrations above  and  below the EC-10
and EC-50 values for algal growth  and mortality.   If the
chemical has no measurable effect  at the  saturation
concentration (at least 1000 rng/1)/  it is considered
relatively nontoxic to algal growth  and definitive testing
for effects on these processes is  deemed  unnecessary.  In
all cases, the range-finding test  is conducted  to  reduce the
expense involved with having to repeat a  definitive  test
because of inappropriate test chemical concentrations.
         3.  Definitive Test
    The specific requirements of  the definitive test are the
analytical determinations of chemical concentrations, the
unbiased selection of algae for each treatment, the  use  of
controls, the assessment of test  validity, and  the

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recording, analysis, and presentation of data.  These
requirements assure that the chemical concentration  -  algae
response relationship is accurately known, that chemical
effects are not confounded by differential algal  growth  and
that the relationships are clearly present.   Reporting the
occurrence of such abnormal effects as  irregular  cell  size
or shape, clumping, loss of chlorophyll, cell mortality,  or
other unusual effects provides qualitative data that further
assist the assessment of phytotoxicity.
    The purpose of the definitive  test  is  to  determine the
EC-10, EC-50 and jconcentra.tion-response  curves for algal
growth for each species tested with a minimum of  testing
beyond the range-finding test.  The concentration range  for
the definitive tes c is based upon  tne results of  the range-
finding for that species.  It is probable  that each  of the
species tested may have a different estimated EC-50  based on
the range-finding  test and that more than  five
concentrations of  a test substance in a  geometric series  may
be needed to properly describe 'he dose-response
relationship for either species being tested.  By testing a
minimum of five concentrations in  a series per species
the dose-response  relationship will oe  better defined.   The
slope and shape of the dose-response curve can give  an
indication of the  mode of action of the  chemical  and will
allow estimatnon  of the effects of lower  concentrations  on
the algae.
    The primary observations - number of  algae per  chemical
and determination  of the actual chemical  concentrations
employed  in  the definitive test, are needed  to accurately
describe  the dose-response curve from which  the EC-10  and
EC-50 are calculated.

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    The recommended experimental  design  is  the randomized
complete block.  As discussed  by  Hammer  and Urquhart (1979),
it is essential that the  investigator  randomly assign test
containers to  treatments  to  assure  that  each aliquot of
algae has the same chance of receiving any  of  the  treatments
(exposure level of test chemical).   To account for variation
within the growth chamber and  to  increase the  sensitivity
for detecting  treatment differences, small  square  blocks
should be delineated in the  growth  chamber  with
randomization  of treatment within blocks.   Replication
should occur over growth  chambers (of  the same type)  as,  in j
many cases, a  wi thin-growth  chamber estimate of residual
variance badly underestimates  the between chamber  estimate
(Hammer and Urquhart 1979).  This means  that differences
between growth chambers are  often greater than differences
between growth and environmental  conditions within chambers.
         4.  Analytical Measurements
    The actual chemical concentration  used  in  the  definitive
test should be determined with the  best  available  analytical
precision.  Analysis of stock  solutions  and test solutions
just prior to use will minimize problems with  storage (e.g.,
formation of degradation  products,  adsorption,
trans for-nat ion, etc.).  Nominal concentrations are  adequate
for the purposes of the range-finding  test.   If definitive
testing is not required because,the  chemical elicits  an
insufficient response at  the 1000 mg/1 level  in the range-
finding test,  the concentration of  chemical  in the  test
solution should be determined  to  confirm tne actual exposure
level.

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    The pH of the test solution should be measured  prior  to
testing to determine if  it  lies outside of  the  species
optimal range.  While it is recognized that  algae may grow
over a broad range of hydrogen-ion concentrations and
typically exhibit a pH optima  for logarithmic growth, this
,test guideline does not  include pH adjustment for the
following reasons: the use  of  acid or base  may  chemically
alter the test substance making it more or  less toxic,  the
amount of acid or base needed  to adjust the  pH  may  vary from
one test solution concentration to the next, and  the effect
the test| chemical has on pH may indirectly  affect growth  and
development  of the algae.   Therefore, the pH of each test
solution should be determined  and compared  to the acceptable
range for growth and development of  the test algae.
    The data obtained in bioassays are usually  expressed  as
standard response curves in which growth response of tne
test species is plotted  against the  concentration of the
test chemical.  The manner  of  expressing algal  growth
response varies cons ideraoly.  For this guide "me algal
growth responses are expressed as direct measurements  of
number of algae per ml of solution.  The statistical
analysis (goodness-of-f it determination) facilitates
accurate calculations of EC-10 and EC-50 as  well as
providing confidence limits for the  concentration  (dose)-
response curve.
    B.  Test Conditions
         1.  Test Species
    Both Salenas trum  capr icornu turn and  Skele tonema cos tatum
have a  number  of  useful  characteristics  as  listed  below,
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which are necessary  for  an  algal  species to be used in
bioassays (Toerien et al.  1971):
     (a)       broad  nutrient  response (grows both in
              oligotrophic  and  euthropic waters).
     (b)       distinct shape
     (c)       uniforra s ize
     (d)       divide  distinctly
     (e)       do  not  attach to  glass or surface
     (f)       stay in suspension  with slight agitation
     (g)       cells  do not  clump  (aggregate)
     (h)       grow at a  maximum rate in a short tme in a
              medium  simple to  constitute
     (i)       do  not  excrete  autotoxins
     (])       cells  are  easy  to count by both direct or
              indirect methods.
     Selenastrum capr icornu turn is  an excellent laboratory
freshwater organism,  easy  to  culture and count, and is both
sensitive and consistent in its response to a wide range of
nutrient levels  (Payne and  Hall 1979).
    When included in  multispecies  toxicity screening tests,
Selenastrum has been  found  to be  a comparably sensitive
species.  Maki and Macek (1978) found this to be true in an
environmental safety  assessment for a nonphosphate detergent
builder.  Selenastrum was as  sensitive  to trinitrotoluene as
the copepod, Trigriopus  californicus,  and was  twice as
sensitive as oyster larvae  (Smock  et al. 1976).  Selenastrum
was as sensitive as Daphnia and the fathead minnow to eight
preparations of synfuels  (Greene,  personal communication).
In a study of the toxicity  of 56 dyes  to Selenastrum and
fish (fathead minnows),  basic dyes do not markedly inhibit
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algal growth, and "of special significance, however,  is  the
rather startling correlation between  results  of  algal  assays
and the results of fish bioassays"  (Little and Chillingworth
1974).  Greene (personal communlation) analyzed  the  results
of this study and found the algae appear  -nore sensitive  than
fish to 35 of (the dyes tested while the fish  were  only more
sensitive to seven of the dyes  tested.  In a  recent  test
conducted on 35 chemicals on the  EPA  priority pollutant  list
by EG & G Bionomics  (Parrish, personal communication), there
were no significant  differences in  the EC-O's between
Serenastrum, and Skelet;onema, Daphnia  and  bluegill  fish,
Lepomis macrochirus.  Selenastrum was significantly  more
sensitive than sheepshead minnow.   In another 2  tests  EG & G
performed for Monsanto Industrial Chemical Co.  (1979a,b)
evaluating two phthalate esters (Santicizer 60 and 711),
Selenas trum was as sensitive as i4icrocystis aerugenosa,
Navicula pelliculosa, Skeletonema costatum and Dunaliella
tertlolecta.  Palmer (1969) has extensively reviewed the
algal literature and  has ranked the 60 most pollution
tolerant genera as reported by  165  authors.   In  comparing
two  green algae often used  in algal toxicity  testing,
Chlorella and Scenedesmus to Selenas trum, great  variation is
found.  Of the 60 genera, Scenedesmus was the  fourth most
tolerant, Chlorella  was  the fifth most  tolerant, but
Selenas trum was the  fifty-seventh most tolerant.  This
analysis is borne out by recent results obtained by Green
(personal communication) in testing effluent  toxicity to
algae.  He found  that Chlorella and Scenedesmus  are
generally more resistant to industrial effluents and both
were naturally present  in 100%  effluents  (eight  submitted by
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 the  USEPA Industrial Environmental Laboratory, Research
 Triangle  Park,  Raleigh,  North Carolina).  Selenastrum only
 grew when the effluents  were diluted to 1-10% of the
 original  concentration (which supported Chlorella and
 Scenedesmus  growth).  This was also the case in another
 effluent  which contained 1.7 mg/1 cyanide.  Both Chlorella
 and  Scenedesmus  grew in  it, but Selenas trum grew only when
 the  effluent was diluted to 1% or less.  Chlorella has also
 recently  been shown to be much less sensitive to toxics than
 Daphnia or fish (Kenaga  and Molenaar, 1979).
i   I  While it is  recognized that numerous narine algae are
l  i i                                            '
 sensitive to toxicants (North et al. 1972);  neavy metals
 (Davies 1978),  simple organics (benzene, cresol, hexane,
 phenol  and toluene), various inorganics (Cl, CN, Hg)  and
 complex wastes  (industrial sewage, sulfite waste liquor,
 detergent),  and  petroleum compounds (Corner 1978),
 Skeletonema  costaturn was  selected for use in the toxicity
 test guideline.   This species has been frequently reported
 on in the bioassay literature (US Army 1978), and is  a
 recommended  bioassay organism (APH 1975, US EPA 1977a, b,
 1978, Gentile and  Johnson 1974).
     The testing  procedure for Skeletonema has recently
 proven  useful for  the evaluation of the relative potential
 hazards of a compound or a complex waste by  providing data
 for  the calculation of the EC-50  or SC-20 (Walsh and
 Alexander 1980,  Walsh et al. 1980).  Skeletonema was  as
 sensitive to the 35 priority pollutants and  two phthalate
 esters  as Selenas trum in multi-species toxicity screening
 tests,  as in the previously described studies.
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    Skeletonema vas found to be tiore sensitive (at lOppb) to
growth inhibition effects induced by PCB's than two
freshwater algae (Euglena gracilis and Chlamydomonas
rein'nardt 11) and two other marine algae (Thai ass IPS ira
pseudonana, and Dunaliella tertiolecta) (Mosser et al.
1972).
    Skeletonema costaturn was also more sensitive  (growth
inhibited) at lower concentrations of wastewater
chlorination products ( 3-chlorobenzoic acid, 5-chlorouracil,
4-chlororesorcinol, 3-chlorophenol and Captan) than
Dunaliella tertiolecta and Porphyridium sp.  (Sikka and    j
Butler 1977).
    Skeletonema and Selenastrum are specified for testing
toxicity of pesticides (Subpart J, Pesticide Registration
Guidelines).  Additional justification for selection  of
these test species is provided in these guidelines (see FR
45  (214):  72948-72978).
    Other  species  may be substituted for either of these  two
species  when appropriate.  Some freshwater or marine  species
which are  of concern or have a significant ecological role
may constitute a more crucial risk population.  If so, those
species  of particular ecological or economic value should be
selected.  The rationale for selection of alternative
species  should be  discussed with the Agency  and/or supported
in  the report of findings.
          2.  Facilities
              a.   General
    The  test requires a growth chamber or temperature
controlled enclosure capable of maintaining  a uniform
temperature of 24 + 1C if Selenas trum  is tested or
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20 +_ 1C if Skeletonema  is  tested.   Other  facilities
typically needed  include  standard  laboratory glassware,
culture flasks, work areas to clean  and  prepare  equipment
and to measure chemical concentrations  and  algal growth  and
proper disposal facilities.  Without these  facilities, the
testing cannot be adequately conducted.
              b.  Test Containers
    Sterile Erlenmeyer flasks are  recommended as test  and
culture containers.  Any  flask volume may be used between
125-500 ml.  However, it  is  imperative  tnat flasks of  the
same volume be used throughout the test.  Hannon and        ,
      I  I i    i  , i r                                          '
Patouillet (1979)  found a marked difference (2.6x)in mercury
toxicity for narine algae, Phaeodactylum tricornutum,
depending on the surface  : volume  ratio  of  the culture
vessel.  Flasks should be stoppered  with sterile plugs (such
as foam rubber or cotton  stoppers) which will prevent
possible bacterial contamination yet allow  air flow.
              c.  Cleaning and Sterilization
    Standard good laboratory practices  are  recommended to
remove dust, dirt, other  debris, and organic and inorganic
residues from the test containers  and otner glassware  and
supplies should be washed and sterilized to prevent
contamination.
    Algal cells are discarded at the end of  a test.  Algae
are capable of  considerable  adaptation  to the toxic effects
of antimetabolites and antibiotics,  such as  streptomycin,
penicillin, chloramphenicol, sulfanilimide  and sodium
selenate (Kumar 1964).
    It is important to avoid contamination  of algal cultures
by bacteria.  Bacteria may metabolize high  molecular weight
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organic compounds to produce carbon dioxide and/or  cofactors
that stimulate growth of Selenastrum  (Tison and  Lingg  1977,
Sachdev and Clesceri 1978).  Consequently  axenic  cultures  of
algae should be maintained by proper  sterile  culture
techniques as well as growing and testing  algae  in  sterile
containers and nutrient medium.
              d.  Conditioning
    Test containers are to be rinsed  with  appropriate  test
solutions prior to the beginning of the  toxicity  tests.
This method should allow for sorption of the  test substance
to the test container, thereby,saturating  the container
burface so that no further interactions  of test  substance
will take place when new test solution is  added  and the  test
begins.  Hannan and Patouillet  (1979)  found  that up to 50%
of mercury could be lost to adsorption to  vessel  walls in a
two-day toxicity test.  Therefore,  with proper conditioning
all the test suostance in the test  solution should  be
available to test algae and any results  will  reflect  an
accurate concentration response.
              e.  Nutrient Medium
    The nutrient medium recommended  in the test  guideline,
are those currently recommended by  the USEPA  for  use  in
bioassays (USEPA 1977, 1978a,b,c, Walsh and Alexander  1980,
Walsh et al. 1980) .
    Use of the  nutrient media under the  test  conditions  will
ensure maximum  growth rates (i.e.,  logarithmic)  in  test
algae and controls.   Selenas trum  and  Skeletonema  will  divide
2-3 times per day  (Nielsen 1978,  Lewin and Guillard 1963,
USEPA 1971b).   This should enhance  exposure of test algae to
the test substance because algal cells in  this growth  phase
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absorb and metabolize suostances  at  a  rapid  rate  (Fogg
1965).  Shiroyama et al.  (1973) found  maximum phosphorus  and
nitrogen uptake occurred  in  the first  five days of  growth.
    rtany media used for culturing  algae contain a chelating
agent, usually EDTA, to keep micronutrients  in solution.
However, a medium containing a chelating  agent is less  than
ideal for testing toxicants  because  chelators  can increase
or decrease toxicity and  can add  uncertainty to the test
results (Payne 1975, Fogg 1965, Prakasn and  Rashid  1968,
Bender 1970, Giesy 1974,  Lin and  Schelske 1979, Barber  and
Ryther 1969, Johns ton , 1964, i  Droop  1960,  1962;  Eyster  1968,
                    I  I i    i  i i i
Erickson et al. 1970).
         3.  Environmental Conditions
    Selenas trum and Skeletonema will  grow over  a  wide
temperature range, from less than  5C to 35C  (Claesson  and
Forsberg 1978), and between 13C and  30C (Fogg 1965),
respectively.  The temperature selected for  toxicity testing
using Selenas trum was 24C because  luxury uptake  of ammonia
nitrogen, maximum specific growth  rate, and  sensitivity  to
phenol occur at that temperature (Reynolds et  al.  1974,
1975a, 1975b 1976).  The test temperature 20C  selected  for
Skeletonema is recommended in other toxicity testing manuals
(USEPA 1978a,c) and in recent publications (Walsh  and
Alexander 1980, (Walsh and Alexander  1980, Walsh  et al.
1980).
    Algae require light for photosynthesis and  growth.
Fitzgerald (1975) and Miller et al. (1978) have shown that
light intensity will affect the rate  of growth  of
Selenastrum.  As practically all the  provisional  algal assay
procedure (Joint Industry/Government  Task Force 1969)
                                17

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                                                        ES-5
                                               August,  1982
development work was done on Selenastrum at 400 ft-c,  it  was
not seen as necessary to make a change  (USEPA  1978b) .
Continuous lighting of algal cultures is required  for
Selenas trum in the  test guideline.  While  this does  not
reflect environmental conditions, it does  maximize testing
for toxicity.  Practically all toxicity tests  using
Skeletonema have recommended split day/night lighting  (USEPA
1978a, 1978c, Walsh and Alexender 1980, Walsh  et  al.
1980).  For the sake of consistency, it was not seen as
necessary  to make a change in the procedure.
    The. test guideline requires a test  solution pH of  7.5
for Selenastrum because it maximizes growth.   Selenastrum
grows between pri 4  and 10  (Brezonik et  al.  1975)  and  -
maximally  between pH 7 and 9.6 (Claesson  and Forsberg
1978).  Maximum adenosine triphosphate  (ATP) (i.e.,  energy
production) occurs  in Selenas trum cultured  between pH  7.5
and 8 (Brezonik et  al. 1975).  The pH selected for testing
with Skeletonema, 8.1, was selected because  it is
recommended by other toxicity testing manuals  (USEPA 197 8a)
and in recent publications (Walsh and Alexander 1980,  Walsn
et al. 1980) and approximates the natural  oceanic pH.  The
pH should  be adjusted as exactly as possible to the  test pH
because fluctuations in pH affects toxicity.
    The purposes of oscillating the cultures are  to  enhance
exposure of algal cells to test suostances  and to enhance
dissolution and solubiliza tion of teb t  substances in the
test solution.  Turbulence created by shaking  algal  cultures
is important to enhance the  transfer of dissolved substances
between the nedia and the cells.  Munk  and Riley  (1952)
showed that this transfer  is faster  if  nutrients  are
                                18

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                                                        ES-5
                                                August, 1982
continually renewed  adjacent  to  the  cell by movement of the
medium.
    Oscillating  test containers  is  also analogous to wind
and wave induced  mixing  of  natural  waters.   This agitation
and mixing serves  to maximize algal  exposure to the test
substance.
    Temperature,  light  intensity, pH and oscillation rate
are all recorded  as  specified in the test guideline to
ensure that the  environmental conditions of the test are
me t.
    Temperature  should  be recorded  at least |h'ourly to ensure
that it does not  exceed  the specified limits.   Inexpensive
growth chambers  are  available which  are equipped with
adequate recording ' ins truments or chambers  may be equipped
with ones at minimal  cost.  Severe  fluctuations in
temperature may affect algal  growth  and/or  subsequent
chemical uptake  or metabolism.
               i
    Light intensity  readings  at  the  surface of the solutions
may be made manually  and ensure  that all containers  are
receiving equal  light.   Light variations will affect algal
growth so daily  recordings  are necessary to maintain uniform
and constant radiation.  The  pH  is measured at the beginning
and end of the test  as an indication of effects of test
chemical additions and subsequent algal metabolism on the
hydrogen-ion concentration.   This will  indicate if the test
solution is outside  of the  algal pH  optima  for growth as
well as show what pH  variations  may  exist between chemical
concentrations .
                                19

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                                                        ES-5
                                               August,  1982
    C.  Reporting
    The sponser should submit to the Agency all data
developed during the test that are suggestive or predictive
of phytotoxicity.  If testing specifications are followed,
the sponsor should report that specified procedures were
followed and present the results.  If  alternative  procedures
were used instead of those recommended  in  the test
guideline, then the protocol used should be fully  described
and justified.
    Test temperature, chemical concentrations,  test data,
concentration-respons,e curves, and statistical analyses   ^
snould all be reported.  The justification for  this body  of
information is contained in this support document.  If  algal
species other than tne two recommended  were used,  the
rationale for the selection of the other species should be
provided.
III.   Economic Aspects
    The Agency awarded a contract to Enviro Control,  Inc.  to
provide an estimate of the cost  for performing  an  acute
toxicity test using freshwater algae according  to  the
Guideline.  Enviro Control supplied two estimates;  a
protocol estimate and a laboratory survey  estimate.
    The protocol estimate was $1760.   This estimate was
prepared by identifying the major tasks needed  to  do  a test
and estimating the hours to accomplish  each task.
Appropriate hourly rates were tnen applied to yield a total
direct labor charge.  An estimated average overhead rate  of
115%, other direct costs of $400, a general and
administrative rate of 10%, and  a fee  of 20% were  then added
to  the direct labor charge to yield the final estimate.
                                20

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                                                        ES-5
                                                August, 1982
    Environ Control estimated  that differences in salaries,
equipment, overhead costs  and  other factors between
laboratories could result  in as  much as  50% variation from
this estimate.  Consequently,  they estimated that test costs
could range from $878  to $2636.
    The laboratory survey  estimate was $1465,  the mean of
the estimates received  from eight  laboratories.   The
estimates ranged from  $430 to  #3600 and  were based on the
costs to perform the test  according to the Guideline.
    Altnough a cost analysis was  not performed for a test
using marine algae, the procedures  used  are similar, to thej j
freshwater algal test  and  the  costs should be  similar.
                                21

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                                                        ES-5
                                               August,  1982
IV.  References
APHA.  1975.  American Public Health Association,  American
Waters Works Association, and Water Pollution Control
Federation.  Standard methods for  the  examination  of water
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Barber RT, Ryther JH.  1969.  Organic  chelators  factors
affecting primary productivity in  the  Cromwell  Current
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Bender ME.  1970.  On the significance of metal  complexing
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      !      i
Bold HC and Wynne riJ.  1978.  Introduction to the  algae.
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Brezonik PL, Browne FX,  Fox JL.  1975.   Application of  ATP
to plankton biomass and  bioassay studies.  Water Res.  9:155-
162.

Claesson A and Forsberg  A.  1978.   Algal  assay  procedures
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Corner EDS.  1978.  Pollution studies  with marine
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Council on  Environmental  Quality.   1979.   Ecology and  living
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Davies AG.  1974.  The growth kinetics of Isochrysis  galbana
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Davis AG.   1978.   Pollution studies with  marine  plankton.
Part  II.   Heavy metals.  Adv. Mar.  Biol.  15:381-508.
                                22

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                                                        ES-5
                                                August, 1982


Droop Mr.   1960.   Some  chemical  considerations in the design
of synthetic  culture media for marine algae.  Botanical
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Erickson SJ,  Lackie  N,  Maloney TE.   1970.   A screening
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Eyster  C.   1968.   Microorganic and  microinorganic
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Fisher  NS and Wurs ter CR.   1973.   Individual and combined
effects of  temperature  and polychlorinated biphenyls on the
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	.  1975.   Factors  affecting  the algal assay
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                                              1 i
Fogg GE.  1965.   Algal  cultures  and phytoplankton ecology.
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Galtsoff PS.  1964.   The  American Oyster,  Grasses trea
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Gentile JH, Johnson  MW.   1974.   Marine phytoplankton  In:
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Giesy JP.   1974.   The effects  of  humic acids on  the  growth
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Scenedesmus obliquus  Kuetz.  Ph.D.  thesis.  East Lansing,
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Hammer  PA and Urquhart  NS.   1979.   Precision and
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Hannan  PJ and Patouillet  C.  1979.  An algal toxicity test
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Fed. 51:834-840.
                                23

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                                                        ES-5
                                               August,  1982


Harding LW and Phillips JH.  1978.  Polychlorinated  biphenyl
(PCB) effects on marine phytoplankton photosynthesis and
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Johnston R.  1964.  Seawater, the natural medium  of
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Joint Industry/Government Task Force on  Eutrophication.
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Kenega EE and Molenaar R J.  1979.   Fish  and Daphnia  toxicity
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Leischman AA, Green JC, Miller WE.  1979.  Biblio'graphy of
literature pertaining to the genus  Selenastrum.   Corvallis,
OR: U.S. Environmental Protection Agency.  US EPA-6 00/9-7 9-
021.

Lewin JC and Guillard R.  1963.  Diatoms. Ann Rev.
Microbiol. 7:373-414.

Lightner DV,  1978.  Possible toxic effects of  the marine
blue-green alga Spirulina subsalsa, on the blue shrimp,
Penaeus s tyliros tr is . J.  Invert. Path.  32:139-150.

Lin KG and Schelske CL.  1979.  Effects  of nutrient
enrichment, light  intensity and temperature on  growth of
phytoplankton from Lake Huron.  Duluth,  MN: U.S.
Environmental Protection Agency.  EPA-600/3-76-075.

Little LW and Ch illingworth MA.   1974.   Effect  of  56
selected dyes on growth of  the green alga Selenastrum
capricornutum.  New York: American  Dye Manufacturers
Institute,Inc., pp.2-14.

Lovell RT.  1979.  Fish culture in  the U.S. Science
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Maki AW and Macek  KJ.  1978.  Aquatic  environmental  safety
assessment  for a nonphosphate detergent  builder.   Environ.
Sci. Technol. 12:573-580.
                                24

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                                                        ES-5
                                                August, 1982


Maloney TE and Miller  WE.   1975.   Algal  assays: development
and application.  STP 57J.   Ph iladelpnia, PA:  American
Society for Testing and  Materials,  pp.  344-354.

Miller WE, Greene JC,  Merwin  EA,  Shiroyama T.   1978.   Algal
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Monsanto Industrial Chemicals  Company.   1979a.   TSCA sec.
8(d) submission 8DHQ-1078-0234.   EG &  G  Bionomics  data on
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	.   1979b. TSCA sec.  8(d)  submission  8DHQ-1078-
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Washington, D.C.: Office of Toxic Substances,  U.S.
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Mosser JL. , Fisher NS, Teng TC, Wurster  CF.   1972.         '
Polychlorinated biphenyls:  toxicity to  certain
phytoplankters. Science  175:191-192.

Munk WH and Riley GA.  1952.   Absorption of  nutrients by
aquatic plants.   J. Mar.  Res.  11:215-240.                 ,'

Murray L, Scherifig J, Dixon PS.   1971.   Evaluation of algal
assay procedures  - PAAP  Batch  Test.  J.  Water  Poll. Contr.
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Nielsen ES.   1978.  Growth  of  plankton  algae  as a  function
of N-concentration, measured by means  of a batch technique.
Mar. Biol. 46:185-139.

North WJ, Stephans GC, Nortn BB.   1972.   Marine algae and
their relation to pollution problems,  In:  Ruivo M, ed.
Marine pollution  and sea life.  London:  Fishing News  (Books)
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O'Brien Py and Dixon PS.  1976.   The effects of oils  and oil
components on algae: a review. Br.  Phycol. 11:115-142.

Payne AG.  1975.  Application  of  the algal assay procedure
in bios timulat ion and  toxicity testing.   In  Middlebrooks
EJ, Falkenborg DH, Maloney  TE, eds .  Bios timula tion and
nutrient assessments.   Ann  Arbor, MI: Ann  Arbor Science
Publishers, pp. 3-28.
                                25

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                                                        ES-5
                                                August, 1982
 Payne  AG  and  Hall RH  1979.   A method for measuring algal
 toxicity  and  its application to the safety assessment of new
 chemicals.   In:  Marking LL,  Kimerle RA, eds.   Aquatic
 toxicology.   ASTM Special Technical Publication 667,
 Philadelphia,  PA: American Society for Testing and
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 Prakash A and  Rashid MA.  1968.  Influence of humic
 suostances  on  the growth of  marine phytoplankton:
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 1974.   A  continuous flow kinetic model to predict the effect
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                                 26

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                                                        ES-5
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Shiroyama T,  Miller WF,  Greene  JC.   1973.   Effect of
nitrogen and  phosphorus  on the  growth of Selenastrum
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Sika HC and Butler GL.   1977.   Effects  of  selected
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Smock LA, Stoneburner DL,  Clark JR.   1976.  The toxic
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Toerien DF, Huang CH,  Radimsky  J,  Pearson  EA, Scherf ig J.
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                                27

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                   1973.   U.S.  Environmental  Protection
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                                28

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Vandermeulen JH and Ahem TP.   1976.   Effect  of  petroleum
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                                29

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