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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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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August, 1982
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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August, 1982
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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August, 1982
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° + 1°C if Selenas trum is tested or
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20° +_ 1°C 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 5°C to 35°C (Claesson and
Forsberg 1978), and between 13°C and 30°C (Fogg 1965),
respectively. The temperature selected for toxicity testing
using Selenas trum was 24°C 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 20°C 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)
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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
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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 .
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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.
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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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August, 1982
IV. References
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