United States
Environmental Protection
Agency
Environmental Research
Laboratory
Gulf Breeze FL 32561
Research and Development
EPA/600/8-87/043 March 1988
&EPA Methods for Toxicity
Tests of Single
Substances and Liquid
Complex Wastes with
Marine Unicellular
Algae
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Errata Sheet for EPA/600/8-87/043
Methods for Tox/c/ty Tests of Single Substances and
Liquid Complex Wastes with Marine Unicellular Algae
Please note that Figure 3 (p. 28) and Figure 6 (p. 41) are transposed. The
figure captions are correct.
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EPA/600/8-87/043
March 1988
Methods for Toxicity Tests of Single
Substances and Liquid Complex
Wastes with Marine Unicellular Algae
Gerald E.Walsh
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF ENVIRONMENTAL PROCESSES AND
EFFECTS
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
\5PL-I6]
ir^et, HOOP
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Disclaimer
This document has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or
recommendation for use.
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Table of Contents
Abstract v
Acknowledgments vi
1 Principles of Aquatic Toxicity Testing with Algae 1
1.1 Introduction 1
12 Growth Curve of Unicellular Algae in Culture 1
1.3 Factors That Influence Growth of Unicellular Algae in Culture 3
1.4 Maintenance of Algal Cultures 7
1 5 Choice of Species 7
1.6 Measurement of Population Density 8
1.7 Living and Dead Cells 9
1 8 Numerical Expression of Effect 9
1.9 Addition of Toxicant 10
1.10 Bioaccumulation 10
1.11 Summary 10
1 12 References 11
2. Toxicity Tests With Marine Unicellular Algae 16
2.1 Introduction 16
2.2 Definitions of Terms 16
2 3 Personnel 17
2.4 Safety 17
2.5 Disposal of Toxicants 18
2.6 Quality Control 18
2.7 Chemical and Physical Properties of Test Substances .... 18
2.8 Chemical Analyses of Test Substances 19
2.9 Equipment 19
2.10 Test Species 21
2.11 Axenic Culture 21
2.12 Growth Medium 22
2.13 Medium for Use in Studies on Toxicity of Heavy Metals .. 23
2.14 Medium for Use with Tests on Liquid Complex Mixtures .. 25
2.15 Fractionation Procedure for Liquid Complex Wastes .... 29
2.16 Test for Living and Dead Cells 29
217 Bioaccumulation 29
218 Documentation and Reporting of Results 29
2.19 References 29
3. Growth Test with Single Substances 31
3.1 Preparation of Toxicant 31
3 2 Toxicant Concentrations 31
3.3 Test Procedure 32
4. Growth Tests with Liquid Complex Wastes 38
4.1 Collection of Waste 38
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4.2 Filtering of Waste 38
4.3 Sterile Techniques 38
4.4 Estimation of Population Density ... 38
4.5 Calculation of Effect Concentration 40
4.6 Procedure for Effects of Whole Waste ... 42
4.6.1 Preparation of Medium ... 42
4.6.2 Procedural Steps . . ... 42
4.7 Procedures for Effects of Inorganic and Organic Fractions . 44
4 7 1 Preparation and Testing of Inorganic and Organic
Fractions 44
4.7 2 Procedural Steps . 46
4.8 Procedures for Effects of Inorganic and Organic Subfractions 49
4.8.1 Inorganic Subfractions . 49
4 8.1.1 Preparation and Testing of the Cation
Subfraction . 50
4812 Preparation and Testing of the Anion
Subfraction 51
4.8.2 Organic Subfractions 53
4 8 2.1 Preparation and Testing of the Subfractions 53
4.9 Procedure for Extraction and Testing of Particles 55
4.9 1 Extraction of Organics 55
4.9 2 Testing of Extract 55
4.10 References 55
5. Bioaccumulation Test 60
6. Enumeration of Living and Dead Cells .... 63
6.1 References 64
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Abstract
This manual describes methods for laboratory toxicity testing with marine
unicellular algae. It consists of six parts: Part I describes principles of aquatic
toxicity testing with algae It describes algal growth curves and factors that
influence algal growth including light, temperature, composition of culture
medium, and pH. Methods for maintenance of algal cultures, choice of
species for testing, estimation of population density, detection of living and
dead cells, expression of toxicant effect, and bioaccumulation are discussed.
Part II defines terms related to algal toxicity testing, describes equipment
needed for algal toxicity tests, and gives detailed methods for preparation of
algal growth medium and estimation of population density by cell counts and
spectroscopy.
Part III describes the recommended toxicity test with single substances.
The test is conducted for 48 h, at which time population densities of control
and treated cultures are measured and the median effect concentration and
growth rates are calculated
Part IV gives methods for estimating effects of liquid complex wastes on
growth of algae Methods are described for analysis of whole waste, its
organic and inorganic fractions, anion, cation, base neutral, and acid
subtractions, and particulars matter
Part V describes a method for estimation of bioaccumulation of single
substances by algae in culture.
Part VI gives a method for distinguishing living and dead algal cells
Each of the above tests addresses a different aspect of hazard evaluation
of single substances or mixed waste with regard to inhibition or stimulation of
algal population growth, survival, and possible introduction of toxic substances
into food chains. When population growth tests are combined with studies on
bioaccumulation and algicidal or algistatic properties of a toxicant, an
overview of important effects is obtained, and several relevant effects criteria
provide a base for regulation of single substances and mixtures of
substances.
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Acknowledgments
Sincere thanks are given to the following who reviewed the entire
manuscript of this manual and offered valuable suggestions for its
improvement: Dr. Dennis Ades, Department of Environmental Quality, State of
Oregon, Portland, Oregon; Dr. John H. Duffus, Heriot-Watt University,
Edinburgh, Scotland; Mr. Terrence A. Hollister, EPA, Region VI, Houston,
Texas; Dr. Niels Nyholm, Water Quality Institute, Horsholm, Denmark; Dr.
Ronald L. Rashke, EPA, Region IV, Athens, Georgia; and Dr. Jerry Smrchek,
EPA, Office of Toxic Substances, Washington, D.C.
Thanks also are given to Mrs. Val Coseo, who had the onerous task of
typing several drafts of the manual and did so in a professional and cheerful
manner, and to reviewers at the Environmental Research Laboratory, Gulf
Breeze: Dr. Thomas W. Duke, Dr. David A. Flemer, Mr. Larry Goodman, Mr.
Jack I. Lowe, Dr. Foster L. Mayer, Mr. P.R. Parrish, and Mr. Marlin E. Tagatz.
VI
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1. Principles of Aquatic Toxicity Testing with Algae
1.1 Introduction
This manual describes methods for use of marine unicellular algae in toxicity
tests. Although no common method has been used by testing laboratories,
those described here have been used successfully in tests with pesticides,
heavy metals, other toxic substances, and complex industrial wastes.
Methods have differed mainly in composition of algal growth medium, test
species, and endpoint, each of which may affect expression of toxicity. The
methods described here recommend a carefully formulated growth medium,
sensitive algal species, and three means for estimation of algal population
density.
Toxicity testing with unicellular algae requires application of the principles
of phycology and microbiology to culturing, handling, and exposing the
organisms. Success in culturing and testing depends upon the knowledge
and competence of the responsible person The technician who works with
unicellular algae must be familiar with methods for maintenance of
nonbacterized cultures and their transfer to fresh media, identification of algal
species and deviations from normal morphology, rates of growth of algal
species in culture, use of laboratory instruments, such as microscopes,
counting chambers, analytical balances, light and fluorescence
spectrophotometers, and all other aspects of culture and testing described
below
1.2 Growth Curve of Unicellular Algae in Culture
Algal population growth in culture follows a predictable course under
controlled conditions of light, temperature, and nutrient concentration After
inoculation of growth medium with a known number of cells of a standard age,
the usual pattern of population growth includes a lag phase, phase of
exponential growth, phase of declining growth rate, stationary phase, and
death phase (Fig. 1). See Kaufmann (1981) for a discussion of use of growth
curves The period of time for each phase under optimal conditions is
specific for each algal species. It is not necessary to use synchronous
cultures in toxicity tests because the exponential growth phase, in which cell
number increases at a high rate, is required
The pattern of population growth is very important in toxicity testing
because toxicants and growth stimulating substances cause deviations from
normal (Fig. 2). Generally, two responses of algal populations to test
substances are measured: (1) Rate of growth over part or all of the
exponential phase is determined and growth of treated cultures is compared
with growth of control cultures. Population density or a mathematical
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Logarithm of Population Density
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expression of the growth rates of control and exposed cultures at a selected
time may be compared (2) Final population density is an important criterion
for detection of growth-inhibiting and growth-stimulating substances and for
characterizing eutrophic natural waters.
Length of the lag phase may be critical to success because it can
determine duration of the toxicity test. To minimize the length of the lag
phase, cultures must be initiated with the proper number of cells from algal
stocks in the early exponential phase. Use of older cells may cause a lengthy
lag phase.
Duration of the toxicity test is very important. Algal tests should be
conducted within as short a time period as possible to minimize problems
with fate of the test chemical in the exposure flasks. When the solution, with
algae, is placed under lighting required for algal growth, concentration of the
chemical may decline due to photodegradation, molecular instability,
adsorption to vessel walls, volatilization from the surface of the medium,
adsorption to algal cells, absorption by algal cells, or biodegradation. Shape
of the growth curve in response to a chemical may be due, at least in part, to
behavior of that chemical in the test system For example, loss of chemical
over a selected period of time could determine the rate of decrease in growth
or period of increased lag phase.
Because of the confounding factor of chemical fate, it may be difficult to
interpret algal toxicity data from static tests. Walsh (1983) demonstrated that
the IC50 (interpolated concentration of a toxicant that would inhibit algal
population growth by 50%) increased with time of exposure. Thus, it is
recommended that test duration be no longer than three days, at which time
population densities (which are low) and growth rates (which are high) of
control and exposed populations may be compared by either graphical inter-
polation from density data or by comparison of the growth rates.
If the final yield of algae is required for growth stimulation studies, the
test must be continued until the early stationary phase.
1.3 Factors that Influence Growth of Unicellular Algae in Culture
There are five major factors that affect growth of algae in culture: light
(quality, intensity, duration), temperature, and composition, salinity, and pH of
the growth medium. Each must be controlled carefully, with little deviation
among tests.
LIGHT Algae are cultured under artificial illumination in the laboratory,
but such illumination may differ greatly from natural sunlight in quality
(spectral composition), intensity, and duration These three characteristics
may be regulated for optimal growth of individual species When species with
different optimal requirements are held in a single growth chamber, average
conditions that support growth can be maintained.
Intensity of light has a strong influence on growth and physiological state
of algal populations (Beardall and Morns, 1976, Hitchcock, 1980; Perry et al.,
1981; Cosper, 1982; Soeder and Stengel, 1974; Gallagher et al., 1984;
Falkowski et al., 1985). Gallagher et al. (1984) reviewed a portion of the
literature on effects of light intensity on rates of growth and photosynthesis,
pigments, the photosynthetic unit, and means of photoadaptation of algae. In
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addition, light intensity may influence release of products of photosynthesis
from algal cells (Fogg, 1965; Fogg et al., 1965; Hellebust, 1965, 1967;
Nalewajko, 1966; Watt, 1966, 1969; Bellin and Ronayne, 1968), thus
increasing the amount of dissolved organic matter in growth medium and,
perhaps, affecting uptake of the test substance
Light intensity may also affect behavior of the test chemical. Rates of
photodecomposition of numerous compounds are related to intensity of light
and, when photodecomposition is rapid, it may be the major factor that
determines toxicity. Kamp-Neilsen (1969) reported that toxicity of copper to
Chlorella pyrenoidosa was related to light intensity.
Care must be taken to insure little variation in illumination of algal cultures
because light intensity can influence growth rate and physiological
characteristics of algal populations and behavior of test chemicals. Intensity
of light in growth chambers must be kept constant for all tests and should not
exceed the optimal for algal population growth. Light intensity should be
recorded daily, and when it falls to 90% of optimal, the lamps should be
replaced.
Spectral composition of light can affect growth and photosynthetic rates
and cellular morphology of algae (Humphrey, 1983). As for other
photosynthesizing plants, energy of visible light in the wavelength range of
400 to 729 nm is incorporated by pigments specialized for absorption of
electromagnetic radiation. The various algal divisions contain different
amounts of the three types of pigments that absorb light: (1) chlorophylls that
absorb blue and red light, (2) carotenoids that absorb blue and green light,
and (3) phycobilins that absorb green, yellow, and orange light. Tungsten
lamps emit light in wavelengths used by algae, but also emit a large amount
of heat that is difficult to control in growth chambers. Fluorescent lights also
emit wavelengths that can be utilized by algae, and "cool white" fluorescent
tubes are used commonly, sometimes in conjunction with tungsten lamps.
Spectral signatures of tungsten and fluorescent lamps can usually be
obtained from manufacturers. Whatever the form of lighting, it is essential
that lamps be replaced before gross changes occur in spectral signature.
Replacement when intensity diminishes is probably adequate for consistent
spectral output, but changes in color of light can sometimes be detected
visually. If a color change is seen, the lamp should be replaced.
Marine unicellular algae may be cultivated and tested under continuous
light or under dark-light cycles. Humphrey (1979) described photosynthetic
characteristics of algae grown under both types of regimes' algae grown
under a 12 h light - 12 h dark cycle had a higher photosynthetic rate and a
higher photosynthesis respiration ratio, but a lower growth rate than those
grown under continuous lighting These findings are important to culture and
testing of algae. All stock algal cultures and all tests should be maintained
under the same lighting conditions to insure comparability of results. In
addition, although temperature change has little effect on photochemical
processes, optimal temperature for population growth may vary with light
intensity: increase in temperature increases the point at which light saturation
occurs (Soeder and Stengel, 1974). The test recommended here requires
continuous lighting at constant temperature to insure population densities
large enough to allow statistical evaluation of toxicity data after two days.
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TEMPERATURE Each algal species has a specific culture temperature
for optimal growth at a given light intensity Soeder and Stengel (1974)
reviewed algal adaptation to and effects of temperature on population growth
rate, cell growth, and metabolic processes Algae adapt quickly to
temperature change. However, if tests are to be done at a temperature other
than that at which algal stocks are maintained, then new stocks must be
prepared and adapted to the new temperature. \Temperature should not vary
by more than one degree C from the desired temperature.
ALGAL GROWTH MEDIA There have been numerous reviews of algal
nutrition in relation to composition of growth medium (Provasoli et al., 1957;
Krauss, 1958; Provasoli, 1958, 1966; McLachlan, 1959, 1964; Droop, 1961,
1969; Gerloff, 1963; Nicholas, 1963; Hunter and Provasoli, 1964; Kester et al.,
1967; Venkataraman, 1969; Prat et al., 1972; Healy, 1973; Stein, 1973;
O'Kelley, 1974). Droop (1969) gave 65 references to different growth media
for marine algae. The major thrust in development of marine algal medium
has been elucidation of nutritional requirements and amounts of nutrients and
their ratios required for growth and normal cell morphology. Modern media
are composed either of natural or artificial seawater to which nutrients are
added Nutrient additions include orthophosphate (PGV), nitrate (NOa"),
potassium, magnesium, calcium, sulphur, boron, cnelated trace metals,
vitamins, and silica (for diatoms and some chrysophytes and xanthophytes).
See Droop (1977) for a discussion of nutrition of phytoplankton.
Media made from seawater have been used extensively for culture of
unicellular algae. Guillard and Ryther (1962) described a medium (Medium f)
made from natural seawater that has proved to be excellent for culture of
many algal species. Medium f and its nutritional variations, f-1 (Guillard and
Ryther, 1962) and f/2 (Stein 1973), are still in use today.
Although natural seawater-based media are often excellent for culturing
algae, diel and seasonal variability in its composition may control rate of
growth. Johnston (1962) demonstrated seasonal variability of ocean surface
water to support growth of algae. Media prepared from surface water of the
Gulf of Mexico support growth of algae in our laboratory between October and
April, but not between May and September, even though nutrient enrichments
are identical. Unless a relatively unchanging source is available, toxicity tests
with medium prepared from natural seawater may not yield comparable data
if the seawater is collected at different times.
Growth media prepared from pure chemicals and distilled or deionized
water have been described (Morel et al., 1979; Harrison et al., 1980). See
Morel et al. (1979) for a discussion of requirements for algal synthetic
medium. Such media have the advantage of constant salt and nutrient
composition without possible variations that can occur in natural water. This
is especially important if fluorescence intensity is used for estimation of
population density (Tunzi et al, 1974; Slovacek and Hannon, 1977; Gilbert et
al., 1986). Synthetic media are particularly advantageous for toxicity tests
because there is little batch-to-batch variation: the only significant variables
are the toxicants and their concentrations. In our laboratory, the medium of
Morel et al. (1979) produced good growth with normal cell morphology.
However, medium prepared as described by Morel et al. (1979) is
unacceptable in toxicity tests because algae are very sensitive to the toxicant
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carrier, acetone. We have modified nutrient concentrations slightly to make it
acceptable in toxicity tests.
Algal growth medium can also be prepared from commercial sea salt
mixtures. Preparation of media from commercial sea salts is rapid and easy,
and algae grow well in them when nutrients are added. Although overall
composition is known, there may be lot-to-lot variability, with possible
inhomogeneity within lots of commercial mixtures. We have compared
growth and responses to toxicants in media made from five commercial sea
salt mixtures and natural seawater (Walsh et al., in press). We have used one
product, Rila Salts® (Rila Products, Teaneck, NJ), with good results in many
tests. Although salinity was the same and only small differences were found
in nutrient concentrations, differences in response to the same toxicant were
large. Sensitivity to toxicants was generally greater in medium made from
Rila Salts than other commercial formulations (Walsh et al., in press), with
good growth and normal morphology in carrier controls.
Algal medium must be sterilized before use. Although autoclaving has
been used by many investigators, it is not recommended because of possible
contamination of medium from steam under pressure It is recommended
that basal medium be pasteurized and that nutrients be sterilized by
membrane filtration and added to basal medium immediately before the test.
Responses of algae to acetone when the medium of Morel et al (1979) is
used demonstrate that care must be taken in choosing an algal medium;
although good growth and normal morphology may be obtained with a
medium, that medium may be unsuitable for use in toxicity tests
When liquid complex wastes are to be tested, growth medium is
prepared by addition of salts and nutrients used in synthetic medium (Walsh
and Alexander, 1983; Walsh et al., 1980, 1982; Walsh and Garnas, 1983), and
synthetic medium is used as the untreated control. Filtration should be done
only if algae are present in the waste before testing The medium cannot be
sterilized because physical and chemical properties of the waste may be
altered.
SALINITY Salinity of growth medium affects growth and primary
production of algal populations (Braarud, 1951; Nakanishi and Monsi, 1965,
Vosjan and Siezen, 1968). Algal species may be stenohaline, oligohalme, or
euryhaline. Euryhaline species tolerate a wide salinity range by adjusting
concentrations of organic solutes in the cell (Hellebust, 1976a, 1976b; Ahmed
and Hellebust, 1985), and osmoregulation and osmoadaptation may also rely
on activity and specificity of ionic pumps (Soeder and Stengel, 1974).
Osmotolerant marine algae require time to adjust to hypertonic and hypotonic
stress (Gilmour et al., 1982, 1984), and it is important that algae be cultured in
the salinity of the toxicity test for several stock transfers before the test
begins. Salinity must not change when inoculum culture is added to test
medium.
pH Since growth media and natural seawater are generally around pH 8,
this should not present a problem in culturmg marine algae, but pH should be
measured and recorded before each test. Measurement of pH after a test is
of little value because algal activities may change it by as much as one pH
unit even in the highly buffered medium. Flasks should be shaken gently and
continuously to maintain gaseous equilibrium between growth medium and
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air. This should insure sufficient carbon dioxide in the medium to maintain pH
at less than 8.5.
Deviations from the normal pH can indicate improper preparation of
medium. We have used media between pH 7.6 and 8.2 with good growth and
normal morphology. It is best to use a single medium that is constant from
test to test to avoid effects of pH on toxicity of test substances. However,
when medium is prepared from highly acidic or basic liquid waste, rather than
from deionized water, pH is adjusted to approximately 8.
1.4 Maintenance of Algal Cultures
Stock algal cultures are maintained in the laboratory under conditions of light,
temperature, and growth medium identical to those of the toxicity test.
Inoculation of new medium is done under aseptic conditions during the phase
of declining growth or no later than early in the stationary phase to insure that
healthy cells are transferred. Microscopic examination of stocks should be
made for cellular morphology at each transfer If abnormal cells are seen,
transfers should be made at an earlier time in the growth curve, the culture
may be contaminated, or the growth medium may have been prepared
incorrectly. Usually, abnormal cells are caused by excess of one or more of
the trace metal nutrients Stock cultures are checked for aerobic and
anaerobic bacterial contamination at approximately monthly intervals, or more
frequently if contamination is suspected.
Algal stocks for toxicity tests should be in the early exponential growth
phase to avoid transfer of extracellular algal products and because over 99%
of the cells are living and healthy during this phase (Walsh and Alexander,
1980). The stocks should be examined for cellular morphology and only
normal cells, as found during the exponential phase of population growth,
should be used.
1.5 Choice of Species
The major factor that determines quantitative expression of toxicity is the
species used in the toxicity test. Some species are more sensitive than
others to toxicants, but it is impossible to predict which species, or clone of a
species, will be most sensitive or resistant to a particular toxicant. Choice
should be from ecologically important or abundant, generally sensitive
species that can be cultured in the laboratory and with population growth
rates that allow estimation of density within two or three days of inoculation.
Bonin et al. (1986) listed six principal criteria for choice of test species: (1)
wide geographical distribution, (2) well-known nutrient requirements, (3)
good taxonomical characterization of the strains, (4) small genetic and
phenotypic variability, (5) high growth rate, and (6) ease of handling. They
also presented an important critical review of algae commonly used in toxicity
tests.
When possible, test species should occur naturally in the areas that may
be affected by the toxicant. For example, if a toxicant is a threat to an inshore
environment, a species isolated from a nonpolluted inshore environment may
be used for maximum utility of the test (Murphy and Belastock, 1980).
Usually, algal clones must be purchased from a laboratory that maintains a
culture collection.
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It is usually impractical to maintain more than one or two test species in
culture. Walsh and Alexander (1980) recommended the diatom, Skeletonema
costatum, because it meets most criteria listed above and is generally
sensitive to pollutants. However, estimation of cell numbers cannot be made
on an electronic particle counter because the diatom forms long chains.
Bonin et al. (1986) offered compelling arguments for use of the diatom,
Thalassiosira pseudonana, which occurs as a single cell.
The diatom, Minutocellus polymorphus Hasle, von Stosch and Syvertsen,
is recommended here. It is sensitive to most pollutants, population density
can be estimated easily, and the period of exposure to toxicants is only 48
hours. In addition, estuanne and oceanic clones are available for site-specific
studies.
1.6 Measurement of Population Density
There are three common methods for estimation of population density of
marine algal cultures: cell counts, light spectroscopy, and fluorescence
spectroscopy. Brezonik et al. (1975) reviewed methods for estimation of
population density of cultured algae, Rehnberg et al. (1982) reviewed
limitations of electronic particle counting methods when used in algal tests.
Each method estimates a different aspect of the population. Cell counts give
the number of cells in a volume of medium, and when performed under the
microscope, cell morphology can be examined. Light spectroscopy estimates
the concentration of suspended particles (algae) by absorption of light, and
fluorescence spectroscopy estimates the amount of chlorophyll in cells in
suspension Cell counts and spectroscopy do not give information on cell
size, morphology, physiological state, or if cells are living or dead.
No single method for population analysis is completely satisfactory. For
example, a toxicant may cause a large number of small cells or a small
number of large cells, relative to untreated control populations, with biomass
equal to the control. Such effects lead to false conclusions from all three
methods, unless results are also expressed as cell size, measured with a size
distribution meter on an electronic particle counter, or as fluorescence per
cell. Even here, counts may be misleading because particle counters cannot
distinguish living from dead cells. Fluorescence results may be deceiving
because fluorescence yield is a function of chlorophyll concentration and
chloroplast shape Toxicants may cause change in cell chlorophyll content
without causing change in cell number, or they may cause deformation of
thylakoids, thus affecting fluorescence yield.
Despite problems associated with estimation of population density, we
have found good correlation between results from the three methods and, on
a practical basis, recommend them for use with most toxicants. The
researcher should be aware, however, that some toxicants, notably heavy
metals, cause conformational changes in algal cells that may affect results,
and that effects cannot be reported simply as mathematical expressions
(IC50, EC50, SC20; see below) derived from cell counts or spectroscopal
data. Enumeration of deformed cells may be vital for determination of effects
of some substances.
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1.7 Living and Dead Cells
It is often desirable to identify the mechanism that inhibits algal population
growth. Toxicants may kill cells (algicidal) or simply inhibit the rate of
reproduction (algistatic). Presumably, algicides would have greater effects
than algistats on algae in natural waters because algicides kill resident and
allochthonous species and reduce ability of populations to adapt to the
pollution.
Cnppen and Perrier (1974) described a method for identification of living
and dead cells by direct counts with the vital stain, neutral red, and the mortal
stain, Evans blue. Reynolds et al. (1978) used the method to demonstrate
that chlorine-produced oxidants did not kill algae, and Walsh (1983)
described use of Evans blue in toxicity tests with pesticides. In the method
of Walsh (1983), algae are grown in uncontaminated medium for 48 h and the
living and dead cells enumerated. The algae are then exposed to toxicant for
24 h, living and dead cells again enumerated, and decrease in growth rate
and increase in number of dead cells compared to untreated control
populations. The method provides for rapid estimation of algistatic and
algiadal potentials of toxicants.
1.8 Numerical Expression of Effect
Bioactivity of compounds toward algae is expressed as the interpolated
concentration that would cause a specific effect in a specific period of time.
Thus, the 72-h IC50 is the calculated concentration that would inhibit
population growth or a physiological process of treated cultures by 50% of
untreated cultures in 72 h. The IC50 is also called the EC50 (effective
concentration) Similar expressions are used when cell death is observed
(lethal concentration, LC) or when growth or a physiological process is
stimulated (stimulatory concentration, SC). Test durations have ranged from
hours for physiological and growth endpomts to weeks for growth endpoints,
and effects as percentages of untreated controls have been reported between
1 and 99. Effect concentrations are calculated from tests in which response
to a graded series of toxicant concentrations is measured and percentage (of
untreated cultures) response is related to concentration. Methods that have
been used for calculation of algal response include: graphical interpolation
(Yamane et al., 1984, APHA, 1985), moving average interpolation (Harris,
1959; Stephan, 1977), probit regression (Finney, 1971; Ordog, 1981), and
binomial (Harris, 1959) Walsh et al. (1987) discussed these methods as
applied to growth tests, compared EC50s calculated by them, found no
significant differences, and suggested use of graphical interpolation because
it is simple, rapid, and accurate.
Two other expressions are used to describe toxicity. The Lowest
Observed Effect Concentration (LOEC) is the lowest concentration of a
substance in a toxicity test that has an adverse effect on the exposed
population when compared to the untreated control population. The No
Observed Effect Concentration (NOEC) is the highest concentration used in a
test that has no observed effect on the exposed population.
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1.9 Addition of Toxicant
In toxicity tests, a range of toxicant concentrations is used to encompass the
desired percentage response, e.g , if the IC50 is to be used for expression of
algal response, concentrations should include the NOEC, the 100% response
concentration, and concentrations that inhibit growth by more than and less
than 50%, such as 35 and 65%. At least five concentrations should be used
for establishment of the concentration-response curve.
Most toxicants are poorly soluble in water and must be dissolved in a
solvent carrier before addition to test medium. Concentration of solvent is
critical to success of a test. It must be as low as possible and the same for
every toxicant concentration. Carrier controls must be included in every test,
with algal growth equal to that of noncarrier controls. We have found
(unpublished data) that acetone is less toxic to marine algae than two other
frequently used carriers, tnethylene glycoi and dimethyl sulfoxide. However,
acetone can affect results by interaction with toxicants, and the publications of
Stratton (Stratton et al., 1980, 1982; Stratton and Corke, 1981a, 1981b;
Stratton, 1986, 1987) should be consulted for review of this subject.
1.10 Bioaccumulation
Although not necessarily related to toxicity, accumulation of toxicants from
sublethal concentrations in water may be important to toxicant fate and
transfer through food chains. Rice and Sikka (1973), Hollister et al. (1975)
and Walsh et al (1977) showed that living and dead algal cells can
accumulate toxicants Banner et al. (1977) demonstrated transfer of the
pesticide, Kepone, from contaminated algae to oysters. Bioaccumulation
studies are recommended for a more complete understanding of relationships
between algae and toxicants.
1.11 Summary
Algal toxicity tests include numerous microbiological procedures and culture
criteria that must be controlled carefully for accuracy and reproducibility.
There must be test-to-test consistency in technique to insure comparable
data. Algal stock cultures must be maintained in a healthy, growing state.
Constant monitoring of light intensity and temperature, care in preparation of
growth medium, analysis of growth medium for salinity and pH, and
morphological examination of algal stock, inoculum, and control cultures
should insure healthy populations with reproducible growth rates. Algal
populations used as inocula for toxicity tests must be taken from the early
exponential phase of growth in all tests. By carefully following established
good laboratory practices for culturing and testing, the trained technician can
generate data that describe potential effects of chemical substances on
population growth and survival and on bioaccumulation by marine unicellular
algae.
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1.12 References
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Beardall, J. and I. Morris. 1976. The concept of light intensity in marine
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Bellin, J.S. and M.E. Ronayne. 1968 Effects of photodynamic action on the
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Physiological features of six micro-algae to be used as indicators of
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Braarud, T. 1951. Salinity as an ecological factor in marine phytoplankton
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plankton biomass and bioassay studies. Water Res 9: 155-163.
Cosper, E. 1982. Effect of variations in light intensity on the efficiency of
growth of Skeletonema costatum (Bacillanophyceae) in a cyclostat J.
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Crippen, R.W. and J.L Perrier. 1974. The use of neutral red and Evans blue
for live-dead determinations of marine plankton Stain Technol. 49: 97-
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culture media. Bot. Mar. 2: 231-246
Droop, M.R. 1969. Algae, pp. 269-313, in Methods in Microbiology 3B
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Droop, M.R 1977. An approach to quantitative nutrition of phytoplankton.
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relationships in phytoplankton. Limnol. Oceanogr. 30: 311-321.
Finney, D.J. 1971. Probit Analysis, 3rd Ed., Cambridge University Press,
New York.
Fogg, G.E. 1965. Algal Cultures and Phytoplankton Ecology. University of
Wisconsin Press, Madison.
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phytoplankton photosynthesis. Proc R. Soc. Lond. Ser B 162" 517-
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in the marine diatom Skeletonema costatum influence of light intensity
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Gilbert, P.M., T.M. Kana, R J. Olson, D LKirchman, and R.S. Alberte. 1986.
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199-208.
Gilmour, D.J., M.F. Hipkins, and A. D. Boney. 1982. The effect of salt stress
on the primary processes of photosynthesis in Dunaliella tertiolecta.
Plant Sci Lett. 26: 325-330
Gilmour, D.J., M.F. Hipkins, and A.D. Boney. 1984. The effect of decreasing
the external salinity on the primary processes of photosynthesis in
Dunaliella tertiolecta. J. Exp. Bot. 150: 28-35.
Guillard, R.R L. and J H. Ryther. 1962. Studies of marine planktonic diatoms.
I Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can.
J Microbiol 62: 229-239.
Harris, E.K 1959 Confidence limits for the LD50 using the moving average
angle method Biometrics 15: 424-432.
Harrison, P.J , R.E. Waters, and F.J R. Taylor. 1980. A broad spectrum
artificial seawater medium for coastal and open ocean phytoplankton. J.
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Healey, F.P 1973. Inorganic nutrient uptake and deficiency in algae Grit.
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Hellebust, J.A. 1965. Excretion of some organic compounds by marine
phytoplankton. Limnol Oceanogr 10: 192-206
Hellebust, J.A. 1967 Excretion of organic compounds by cultured and
natural populations of marine phytoplankton. pp. 361-366, in Estuaries
(G H. Lauff, ed.), American Association for the Advancement of Science
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Hellebust, J.A. 1976a. Osmoregulation. Annu. Rev Plant Physiol. 27: 485-
505.
Hellebust, J.A. 1976b. Effect of salinity on photosynthesis and mannitol
synthesis in the green flagellate Platymonas suecica. Can. J. Bot. 54:
1735-1741.
Hitchcock, G.L. 1980. Influence of temperature on the growth rate of
Skeletonema costatum in response to variations in daily light intensity.
Mar. Biol. 57: 261-269
Hollister, T.A., G.E. Walsh, and J. Forester 1975. Mirex and marine
unicellualar algae: accumulation, population growth and oxygen evolution.
Bull. Environ. Contam. Toxicol 14: 753-759.
Humphrey, G.F. 1979. Photosynthetic characteristics of algae grown under
constant illumination and light-dark regimes. J. Exp. Mar. Biol. Ecol. 40:
63-70.
Humphrey, G.F. 1983. The effect of the spectral compositon of light on the
growth, pigments, and photosynthetic rate of unicellular marine algae. J.
Exp. Mar. Biol. Ecol. 66: 49-67.
Hunter, S H. and L Provasoli. 1964. Nutrition of algae. Annu. Rev. Plant
Physiol 15: 37-56.
Johnston, R. 1962. Seawater, the natural medium of phytoplankton. J Mar.
Biol. Assoc U K. 43. 427-456.
Kamp-Neilsen, L. 1969. The influence of copper on the photosynthesis and
growth of Chlorella pyrenoidosa. Dans. Tidsskr. Farm. 43: 249-254.
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Kaufmann, K.W. 1981 Fitting and using growth curves. Oecologia 49:
293-299.
Kester, D.R., I W. Duedall, D.N. Connors, and R M. Pytkowicz. 1967.
Preparation of artificial seawater. Limnol. Oceanogr. 12: 176-178.
Krauss, R.W. 1958. Physiology of the fresh-water algae. Annu. Rev. Plant
Physiol. 9. 207-244.
McLachlan, J. 1959. The growth of unicellular algae in artificial and enriched
seawater media. Can. J. Microbiol. 5: 9-15.
McLachlan, J. 1964. Some considerations of the growth of marine algae in
artificial media. Can. J. Microbiol. 10: 769-782.
Morel, F.M.M., J.G. Rueter, D M. Anderson, and R.R.L. Guillard. 1979. Aquil:
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Rice, C P and H C. Sikka. 1973. Uptake and metabolism of DDT by six
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2. Toxicity Tests with Marine Unicellular Algae
2.1 Introduction
Marine unicellular algae (phytoplankton, microalgae) are essential to normal
function of marine (estuarine and oceanic) ecosystems. As the main primary
producers, algae form the first link in food webs, oxygenate the water, and are
important in cycling of dissolved organic and inorganic substances. It is
important, therefore, to be able to predict possible effects of chemical
pollutants on algal populations with data from toxicity tests on sensitive
species.
The methods given here are static laboratory toxicity tests for detecting
effects of single compounds and complex wastes on growth of laboratory
algal populations, death of algal cells, and determination of bioaccumulation
by algae in vitro. Although specific algal species are recommended, the
method may be used with other species, either unmodified or with slight
variations in light intensity, temperature, or salinity in accordance with their
requirements. The test is not designed to detect fate of toxicants. Effects on
growth and survival in relation to initial concentration of parent compound are
expressed, but effects of possible degradation products, behavior of
chemicals in the test system, or effects upon physical or chemical
characteristics of the growth medium are not.
2.2 Definition of Terms
Unicellular Algae — Members of the Kingdom Plantae whose
individuals are composed of a single cell and whose growth form may be
unicells, chains, or groups. Those used in toxicity tests are phytoplankton
and are known as microalgae in contrast to the multicellular macroalgae.
Axenic Culture — Culture whose living components are known. In
axenic algal cultures, algal species and bacterial contaminants have been
identified. Generally, axenic algal cultures consist of a single species without
bacterial contamination.
Growth Medium -- The liquid in which algae are cultured. It consists of
a basic salt component in water that is similar in composition to seawater and
to which nutrients are added. It may also be made from natural seawater.
Population Growth -- Increase in number or weight (biomass) of algal
cells in a laboratory population. Cell size or morphology may change during
rapid population growth.
Growth Rate -- Change in cell number or biomass of a population over
a specific period of time. This is generally expressed as a positive integer,
but may be negative if a toxicant causes death and disintegration of cells
during the testing period.
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Exponential Growth Phase -- Time of population growth in vitro at
which cell number increases exponentially.
Population Density -- Number of cells in, or weight of, an algal
population at a specific time. In algal toxicity tests, population density is
estimated at the beginning of a test and at one or more times during the
period of exponential growth to allow calculation of the growth rate.
Inoculum Culture -- Stock culture of algae in the exponential phase
that is diluted to a specific concentration of cells and added to test medium
Untreated Control Culture -- Algal culture that is an integral part of
every toxicity test. It is a culture that is prepared, incubated and analyzed as
all other cultures in the test, except toxicant and its carrier are not added.
Carrier Control Culture -- Algal culture that is an integral part of every
toxicity test in which a solvent carrier is used. It is a culture that is prepared,
incubated, and analyzed as all others in the test, except it contains the same
volume of solvent carrier as treated cultures, but without the toxicant.
Rangefinding Test (Screening Test) — Test conducted to determine
concentrations of a toxicant to be used in the definitive test.
Definitive Test -- Test conducted to define the toxic or stimulating
concentration of a chemical. It yields data for calculation of the IC50, LC50,
or SC20.
IC50 (EC50) -- Interpolated or calculated concentration of a toxicant
that would inhibit population growth or any other biological process of algae
by 50% compared to the controls in a specific period of time. This has also
been called the RC50 (concentration that reduces growth by 50%). The term
RC50 is not recommended here because it is an erroneous expression of
effect. Toxicity causes a lower growth rate in relation to control growth: it
does not reduce growth rate in treated cultures.
LC50 -- Interpolated or calculated concentration of a toxicant that
would kill 50% of the cells in a specific period of time.
SC20 ~ Interpolated or calculated concentration of a substance that
would cause population density of a treated culture to be 20% greater than
the controls in a specific period of time.
NOEC -- No observed effect concentration. This is the highest
concentration of a substance that had no observed effect in a toxicity test.
LOEC -- Lowest observed effect concentration. This is the lowest
concentration of a substance that caused an observed effect in a toxicity test.
2.3 Personnel
Personnel designated to perform toxicity tests with algae must be trained
in the principles and techniques of phycology and microbiology. Successful
maintenance of axenic cultures, sterile transfer, identification of deviations
from normal morphology, early detection of problems, and numerous other
aspects of algal culture and testing can be accomplished only by scrupulous
attention to details by knowledgeable people.
2.4 Safety
Safety of laboratory personnel must be insured before initiation of algal
tests with toxic substances and solvent carriers. Manufacturer's data sheets
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and other sources of information on effects of the test material on human
health must be consulted and proper safety measures applied before testing.
Care is especially important when working with liquid complex wastes, where
contents of the mixtures may be unknown. Operators must wear protective
clothing (laboratory coats, gloves, respirators) whenever exposure may occur
to skin or by inhalation, suitable absorbants for carrier and toxicant spills must
be at hand, and the laboratory must be well-ventilated. All liquids must be
transferred by automatic pipette, never by mouth, and flame or sparks must
not be present when solvents are used. Stock solutions must be stored in a
refrigerator equipped with a spark-free motor. Laboratory coats, gloves, and
respirators must be worn when cleaning contaminated glassware with solvent.
When algal tests are done for EPA, each laboratory must have an
approved, detailed, written safety plan that describes methods for safe
handling of toxicants and solvents.
2.5 Disposal of Toxicants
The testing laboratory must have a written plan, approved by EPA, for
safe disposal of toxicant stocks and stock dilutions in solvent, contaminated
growth medium with algae, solvent used for rinsing glassware, and
contaminated equipment such as pipettes, laboratory coats, gloves, and
absorbant used to clean up spills. Disposal will generally require employment
of a commercial company licensed by the State for hauling and disposing of
toxic waste according to RCRA regulations
2.6 Quality Control
The best quality control that can be applied to algal toxicity tests is
related to competent personnel, close adherence to all aspects of the method
as described, and consistency in day-to-day operations. Careful quality
control must be applied to maintenance of algal stock cultures, control of light
intensity and temperature, rigorous cleaning of glassware, a draft-free
working area, application of microbiological techniques for maintenance of
sterility, and periodic testing for bacterial contamination of stocks.
When algal tests are done for EPA, each laboratory must have a detailed,
written quality control plan that describes requirements for culturmg and
testing of algae. A laboratory notebook that records data showing adherence
to good laboratory practices required by EPA should be available for
inspection by the Project Officer.
2.7 Chemical and Physical Properties of Test Substances
Chemical and physical properties of toxic substances and solvent carriers
should be known before the test begins. They can often be obtained from the
manufacturer, seller, or handbooks on such properties, and are used for
setting toxicant concentrations and interpretation of results. Information is
required on solubility, volatility, vapor pressure, rate of decomposition in
water, and octanol-water partition coefficient.
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2.8 Chemical Analyses of Test Substances
Chemical analyses must be performed to confirm identity and purity of
the toxicant and to determine percentage recovery in growth medium before
initiation of algal tests Quantitative analyses must also be done on medium
that contains the concentrations of toxicant used in each test. Identification
and quantitation of major organics, anions, cations, PCV, and NC>3~ in
complex mixtures is also desirable, and results of chemical analyses should
be submitted with test results. When algal tests are done for EPA, the testing
laboratory must have an approved, detailed, written set of protocols for
chemical analyses, including a plan for quality control
2.9 Equipment
GLASSWARE All glassware must be of borosihcate glass or equivalent.
Glassware used for stock cultures and in toxicity tests should be capped with
stainless steel closures or foam plugs. Flasks and closures distributed by
Bellco Glass Co , Vmeland, NJ, work well. Foam plugs may be used only
once because they absorb volatile chemicals. Their use is discouraged
because they must be disposed of as solid waste.
Volume of growth medium used for maintenance of algal stocks should
be limited to an amount easily handled for sterile transfer. It is suggested that
200 ml of growth medium in a 500 ml volume Erlenmeyer flask is a
convenient system because sterile transfer is difficult with larger flasks.
Volume of growth medium should not exceed one-half that of the
Erlenmeyer flask, whatever the volume.
Flasks of 125 ml volume are used in toxicity tests. If heavy metals are to
be tested, the flask should be coated with silicone film such as General
Electric SC-87 dry film (Pierce Chemical Co., Rockford, IL) or equivalent.
The method for silicone coating was described by Davey et al. (1970). Use
2 5 - 5.0% SC-87 in cyclohexane to coat the flasks. Swirl the solution to
cover the flask wall, dry in air, cure at 150 - 175 C for 4 h, rinse with
deionized or glass-distilled water, and dry again. Inspect all flasks after
each test and recoat over the old silicone layer if necessary
Fernback flasks of 2.8 L volume are used in bioaccumulation studies.
It is necessary to use clean flasks that are not scratched inside because
such flasks cause precipitation of salts from growth medium. Medium in all
flasks should be examined for precipitated salts before each test
Glassware must be washed thoroughly to avoid carryover of toxicant from
test to test. When a test is completed, dispense all contaminated algal
cultures to a safe container and rinse three times with acetone into the
container. Rinse the flasks with tap water and soak them overnight in 1N HCI,
scrub in phosphate-free detergent, rinse 10 times with tap water and three
times with glass-distilled or deionized water, dry in an oven, and store
capped in a draft- and dust-free room
GROWTH CHAMBERS Separate growth chambers must be maintained for
algal stock cultures and toxicity tests They must be kept under identical,
carefully controlled conditions of light (quality and intensity) and temperature.
Light should be from cool white fluorescent tubes, with variable control of
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light-dark cycles or continuous lighting. Intensity may vary with algal
species, but is generally between 50 and 150 pE rrv2 sec~1. Intensity may
be controlled by rheostat or by placing cultures at selected distances from the
fluorescent tubes. Light intensity in the tests recommended here may be
between 100 and 125 p,E m-2 sec-1 (590-735 ft can; 6,350-7,900 lux) at
the surface of the growth medium. When light intensity falls below 100 pE
nrr2 sec-1, the fluorescent tubes should be replaced. Intensity of light from
new tubes falls rapidly during the first four days of use and remains constant
for many days thereafter Therefore, it is best if tests are not initiated until
four days after the fluorescent tubes are replaced.
Temperature in the growth chamber may also vary with requirements of
individual algal species. Generally, a chamber that can maintain constant
temperature between 10 and 25 C (±0 5 C) is adequate Temperature in the
test described below is 20 C.
LIGHT METER Light intensity must be monitored at least once a day at the
level of the surface of the growth medium. The light meter must be calibrated
against a standard, and measurements must be recorded daily.
TEMPERATURE MONITOR A device for continuous recording of
temperature must be installed in the growth chamber It should read in °C,
and all original temperature records should be kept in a permanent file.
MICROSCOPE A microscope with resolving power that allows detailed
examination of algal cells is needed for checking cell morphology. It must
have a mechanical stage that accepts a hemacytometer for counting cells.
STERILIZATION OVEN Algal growth medium is sterilized by heat. The
sterilization oven must be large enough to contain all flasks of each toxicity
test and must maintain its temperature at 60 C for 4 h.
AUTOCLAVE An autoclave is used to sterilize everything other than
growth medium. The autoclave must have a drying cycle to insure dry
glassware and filters.
PIPETTORS Medium for toxicity tests is added to exposure flasks with a 50-
ml volume pipettor. The Ace Glass (Louisville, KY) Volumetril Bulb and 500-
ml volume flask work well. Nutrients are added with Eppendorf pipettes (or
equivalent) fitted with disposable tips. Metal and vitamin additions require
sterile tips. Sterile, disposable glass pipettes are required for culture transfers
and sampling.
HEMACYTOMETER A hemacytometer is needed to estimate population
density of inoculum cultures and for enumeration of cells in toxicity tests.
HAND TALLEY A multi-channel hand talley device is required for counting
cells on a hemacytometer and for differential counts of living and dead cells.
ELECTRONIC CELL COUNTER An electronic particle counter may be used
to estimate population density of algae that occur as unicells, but not as
doublets, chains, clumps, etc. It is desirable to have an attachment to the
counter for estimation of average cell volume.
SALINOMETER A hand-held salinometer is required to check salinity of
growth medium and raw liquid waste
VISIBLE LIGHT SPECTROPHOTOMETER Population density may be
estimated with a visible light spectrophotometer equipped with variable
wavelength control and a 10-cm light path. This equipment may be used
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when the only particles in the culture are algal cells, the population is in the
exponential phase of growth, and the medium is not highly colored
FLUORESCENCE SPECTROPHOTOMETER Population density may also
be estimated by fluorescence spectroscopy of intact cells. This method is
very sensitive, especially at low population density.
ROTARY SHAKERS Cultures in algal toxicity tests must be shaken gently to
insure exchange of oxygen and carbon dioxide between the atmosphere and
growth medium, and to maintain the cells in suspension for optimal utilization
of nutrients and light.
pH METER pHs of stock and test media are measured and recorded before
each transfer or test.
CENTRIFUGE A high-speed centrifuge is used in studies on
bioaccumulation. Buckets may be up to 500 ml in volume and lined with
Teflon (E.I. DuPont de Nemours, Newtown, CT) or equivalent, but never
metal.
MAGNETIC STIRRER Magnetic stirrers are used for preparation of growth
medium and resin for cation removal.
2.10 Test Species
Algal species used in toxicity tests may vary in accordance with requirements
and objectives of the tests, and the method given here should accommodate
most marine unicellular algae after slight modifications of salinity or nutrients.
If a specific species is not required, the test species should be
Minutocellus polymorphus Hasle, von Stosch and Syvertsen (1983). This
species, earlier called Bellerochea polymorpha by Margraves and Guillard
(1974), is found in estuaries and the open ocean, is easily cultured, and is
sensitive to toxicants (Fisher et al., 1972; Fisher, 1977). In the United States,
M. polymorpha can be obtained from the Bigelow Laboratory for Ocean
Studies, West Boothbay Harbor, Maine 04755. It is suggested that Clones
675D, BCN, or SD be used in toxicity tests because they are oceanic and
thus tend to be more sensitive to most toxicants than clones from inshore
waters. However, Clone SAY 7, isolated from an estuary, may also be used.
An important consideration for use of M. polymorphus is its rapid growth
in culture. Toxicity tests can be completed after 48 h growth, thus reducing
the influence of photodecomposition, volatilization, and adsorption of toxicant
that may occur in tests of longer duration.
Starr (1971) and Rosowski and Parker (1982) have identified laboratories
from which algal cultures may be obtained.
2.11 Axenic Culture
Axenic cultures of algae are uncontaminated by bacteria or other organisms.
Algal cultures used in toxicity tests should be axenic and tested for the
presence of bacteria at approximately monthly intervals.
The ATCC Media Handbook (1984) gives formulations of media for
detection of bacterial contamination At least two media should be used:
LBacto Peptone 10.0 g
Succmic Acid ... 1.0 g
(NH4)2SO4 ... 1.0g
21
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MgSO4-7H2O 1.0 g
FeCI3-6H20 2.0 g
MnSO4-H20 2.0 g
Synthetic Seawater 1.0 L
2. Bacto-Tryptone Yeast Extract 5.0 g
Glycerol 3.0 g
Synthetic Seawater 750 ml
Distilled or Deionized Water 250 ml
Synthetic Seawater
NaCI 27 5 g
MgCI2 5.0 g
MnSO4 7H20 2.0 g
CaCI2 0.5 g
KCI 1.0 g
FeSO4 1.0 g
Distilled or Deionized Water 1.0 L
Adjust to pH 6.8 with KOH
Dispense 10 ml of medium into test tubes, cap with stainless steel
closures or foam plugs, and sterilize by autoclaving at 121 C for 15 min.
Inoculate the sterile media with 0.25 ml of stock algal culture and incubate at
20 C Examine the media for bacterial growth daily for seven days. After
seven days, streak ahquots of the media on 1.5% agar prepared with the
bacterial media and incubate at 20 C for seven days.
If bacterial contamination is detected, the stock culture must be purified
or a new culture obtained. Techniques for purification are given by Stein
(1975). The easiest method is by treatment with antibiotics, and the
techniques described by Stein (1975) for purification in liquid and on agar
should be used.
2.12 Growth Medium
BASIC MEDIUM The algal growth medium is a modification of that described
by Morel et al (1979). This medium, called "Aquil," is completely artificial
and chemically defined. It is designed to reduce contamination by trace
metals and to contain nutrient concentrations similar to those of natural
seawater, thus precluding complications due to precipitation of salts. Morel et
al. (1979) recommended removal of cations from the major salt and nutrient
solutions by use of an ion exchange column. This step may be used when
the toxicants in question are heavy metals, but it is possible that the ion
exchange process may modify the medium and introduce contaminants.
Untreated medium can be used with all other toxicants. Separate algal stocks
should be maintained in both media.
Formulations of salt and nutrient solutions are given in Table 1. The
sequence of steps in preparation and inoculation of 1 L of growth medium is:
1 Dissolve all salts, except MgCI2-6H2O, in approximately 750 ml of
glass-distilled or deionized water in a 1-L volumetric flask. The water
should have a resistivity of at least 18 megohm-cm at 25 C.
2 When all of the salts have dissolved, add the MgCL2-6H20.
22
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3. When the MgCl2'6H2O has dissolved, add 0.25 ml of each of the
NO3~, and SiOa" nutrient solutions.
4. Bring to 1 L volume with glass-distilled or deiomzed water. Salinity
should be approximately 35 parts per thousand. Check salinity with a
salinometer.
5. Filter the growth medium through a 0.45 jam porosity filter washed
previously with 1 L of deiomzed or distilled water.
6. Dispense into culture flasks. Add approximately 200 ml to each 500
ml volume Erlenmeyer flask for stock cultures. Add 50 ml to each 125 ml
volume Erlenmeyer flask with a volumetric bulb. Dispense approximately 1 L
into each Fernbach flask for bioaccumulation studies
7. Heat sterilize in an oven at 60 C for 4 h.
8. Immediately before the test, mix equal volumes of the metal and
vitamin solution, sterilize by filtration, and add 2 ml to each stock culture flask
with 200 ml growth medium, 0.25 ml to each test flask with 50 ml growth
medium, and 5 ml to each liter of medium in bioaccumulation studies
9. Use a sterile membrane filter (0.45 jam porosity) and non-metallic
filter holder affixed to a sterile syringe for sterilization of the metal and vitamin
mixture. Filter into a sterile capped tube and add to growth medium with an
Eppendorf pipette equipped with a sterile tip.
10. For toxicity tests, add toxicant in no more than 0.025 ml solvent
carrier to medium in exposure flasks; add the same volume to medium in
carrier control flasks All flasks must receive the same amount of solvent
carrier. Reserve other flasks without carrier to serve as untreated controls.
11. Add 1 or 2 ml of old stock at the end of the exponential growth phase
to 200 ml fresh medium for maintenance of algal stocks. For toxicity tests,
add 1 ml of inoculum culture, prepared as described below, to 50 ml growth
medium. These transfers must be done by microbiological methods that
insure sterility.
2.13 Medium for Use in Studies on Toxicity of Heavy Metals
Contaminating cations may be removed from the basic salt and nutrient
solutions before tests for heavy metal toxicity in water. The method for cation
removal was given by Morel et al. (1979). Their report should be consulted
before proceeding with this test, which should never be used with acetone
carrier. A chromatography column, packed with resin that retains cations by
chelation, is used to strip impurities before the salts and nutrients are mixed.
For one column:
1. Use Chelex 100 resin (drymesh 100-200, Na form; Bio-Rad
Laboratories, Richmond, CA).
2. Rinse 15 g resin for 5 min in approximately 200 ml methanol in a
400-ml beaker.
3. Rinse the resin three times in glass-distilled or deiomzed water.
4. Add 300 ml of the solution to be cleaned and, while stirring on a
magnetic stirrer, titrate very slowly to pH 8 (±0.1) with 1N NaOH for the salt
solution and 1N HCI for PO4", NC>3~, and SiOa" solutions. Titrate until pH is
stable for at least 30 min.
5. Fill a 10 x 250 mm glass chromatography column with the solution to
be cleaned A column with a reservoir at the top will save much time. While
23
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Table 1. Composition of Salts and Nutrients to be Used in Culturing
and Testing Marine Unicellular Algae (Morel et al., 1979)
Substance1
NaCI
CaCI2-2H20
KBr
NaF
KCI
H3B03
Na2SO4
NaHC03
SrCI26H2O
MgCI26H2O2
QL-1
24.53
1.54
0 10
0.003
0.70
0.03
4.09
0.20
0.017
11.1
1 The first nine substances are weighed previously to the test. Several batches of dry salts
may be prepared at one time and stored in tightly capped glass containers for use when
needed. Since it is convenient to prepare two liters of medium for each test, batches of
salts that contain twice the amounts given here may be stored and used to prepare growth
medium in glass-distilled or deionized water
2 The MgCI2 6H2O is dried at 104 C, stored in a desiccator, and added only after the
other salts have dissolved.
Nutrients
1.Dissolve 1.38 g Na2HPO4-H2O and 5.26 g NaCI in 1 L glass-distilled or deionized
water.
2.Dissolve 8.5 g NaNO3 in 1 L glass-distilled or deionized water . Ad)ust pH to 8.0 with
INNaOH.
3.Dissolve 3.55 g Na2SiO3 9H2O and 4.38 g NaCI in 1 L glass-distilled or deionized
water. Adjust pH to 8.0 with 1N HCI.
Trace Metals
1.Dissolve 0.47 g Na2EDTA and 0.031 g FeCl3'6H2O in approximately 300 ml glass-
distilled or deionized water in a 500 ml volumetric flask. If heavy metals or liquid waste are
to be tested, use one-half of the iron concentration and prepare this solution without
EDTA immediately before the test
2.Dissolve 0 249 g CuSO4 5H20 in 1 L glass-distilled or deionized water.1
S.Dissolve 0.265 g (NH4)6Mo7O24 4H2O and 0.595 g CoCI2-6H2O in 1 L glass-distilled
or deionized water.1
4.Dissolve 0.455 g MnCI2-4H2O and 0.115 g ZnSO4-7H2O in 1 L glass-distilled or
deionized water.1
24
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the solution is dripping at about 40 ml min'1, pour the resin slurry that was
5.Add 0.25 ml of 2, 3, and 4 to 1 above and take to volume. This if the metals working
mix.
1 Unused portions of these solutions may be frozen for up to a year for later use.
Vitamins
1.Dissolve 110 mg Vitamin 8,2 in 100 ml glass-distilled or deionized water.
2.Dissolve 10 mg biotin in 100 ml glass-distilled or deionized water.
3.Add 0.025 ml Vitamin B12 and 0.25 ml biotin solutions to approximately 75 ml glass-
distilled or deionized water in a 100 ml volumetric flask. Dissolve 0.005 g thiamin
hydrochlonde in it and take to volume. This is the vitamin working mix. Keep frozen when
not in use.
adjusted to pH 8 into it at a constant rate and without interruption to insure a
continuous, homogeneous column of resin.
6. After all of the slurry has been added, continue flow of salt or nutrient
solution until the column is completely packed.
7. Pass the solution to be cleaned through the column at the drip rate of
5 ml min'1.
8. After it has passed through the column, bubble air vigorously through
the solution to return the pH to 8. The air should be filtered through a cotton
plug.
9. Prepare separate columns as above for the salt, PCV, NC>3~, and
SiC>3~ solutions. The metal and vitamin solutions are not stripped of cations.
10. After the pH is returned to 8, medium is prepared as described
above for nonstripped medium.
11. Resin should be replaced after 50 L of salt solution or 100 L of
nutrient solution have eluted. It should also be replaced if it becomes
discolored.
12 The columns may be kept at room temperature when not in use, but
they must not become dry. If the resin breaks, or if any part of it dries out, it
must be regenerated to pH 8 and replaced
It is suggested that several columns for stripping the salt solution be used
simultaneously because this step is time-consuming.
2.14 Medium for Use with Tests on Liquid Complex Mixtures
Follow instructions for preparation of medium as described above, except use
liquid waste instead of water in the salt solution. Do not filter the waste before
adding salts unless algae are present, and do not sterilize. Flasks used in
waste studies should be coated with silicone as described above.
Polycarbonate flasks may also be used. Prepare the metal solutions (Table
1) without EDTA.
Liquid waste must be collected, stored, and shipped in unreactive
containers at 4 C and tested as soon as possible after receipt at the
laboratory. If the waste cannot be analyzed immediately, store it in the dark
at approximately 4 C. Characteristics such as pH, salinity, color, smell, and
presence of solids should be reported with results of toxicity tests.
25
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ESTIMATION OF POPULATION DENSITY Three methods for estimation of
population density are acceptable: cell counts, light spectroscopy, and
fluorescence spectroscopy.
1 .Cell counts -- Estimation of cell numbers may be made by counts on
a hemacytometer. A sample is taken with a sterile pipette after vigorous
swirling of the culture and added to both sides of the hemacytometer. All cells
in the four corner and one central squares of each chamber are counted with
a multi-channel hand tally. The total is the number of cells m a cubic
millimeter of culture medium. This method has the advantage of visual
inspection of algal cells, and those of abnormal morphology may be noted
during the counting procedure. It is, however, time-consuming, relatively
imprecise, and considerable precision is lost as cell numbers decrease.
Photomicrography of hemacytometer preparations with subsequent image
analysis is acceptable for estimation of population density.
Cells may be enumerated on an electronic particle counter. This method
is more rapid and precise than the hemacytometer method, but it can be used
only with species that occur as unicells.
Minutocellus polymorphus occurs as unicells in cultures up to 3 days old.
Cham formation sometimes occurs in older cultures, and electronic particle
counters cannot be used to estimate population density. Since the test
described here requires only two days, chain formation is of little importance.
If an electronic particle counter is used, growth medium, metal mix and
vitamin mix should be filtered through a 0.22 u,m porosity filter before
sterilizing, and an unmoculated blank sample of medium prepared for use in
instrument calibration with regard to background particle interference. This
method is used best with a particle size distribution meter to determine
influence of toxicant concentration on cell size. All cultures should be
examined microscopically for abnormal cells when counts are made on an
electronic particle counter. If appreciable numbers of abnormal cells are
present, estimate their percentage by a differential count of 100 cells. In
order to determine the IC50 as described below, calculate the average counts
for control, solvent carrier control, and each treatment culture.
2.Light spectroscopy — Light spectroscopy is a rapid, efficient way to
estimate population density, provided that the only particles in suspension in
growth medium are algal cells. The spectrophotometer must be calibrated to
zero with growth medium before each test, and checked again for
maintenance of zero calibration after the series of absorbance readings is
completed. The spectrophotometer should be equipped with a recorder for
producing a printed direct readout of results Keep the printed readout in the
laboratory notebook Absorbance of the cultures must be measured across a
10-cm light path to insure precision at low population density and at a
wavelength specific for the algal test species This may be determined by
scanning between wavelengths of 500 to 700 nm and choosing the
wavelength that produces the highest optical density with a culture of algae.
The wavelength is approximately 680 nm for M. polymorphus. Examine the
cultures microscopically for abnormal cellular morphology. If appreciable
numbers of abnormal cells are present, estimate their percentage by
differential counts of 100 cells In order to determine the IC50 as described
26
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below, calculate the average optical density for each control, solvent carrier
control, and treatment group.
3.Fluorescence spectroscopy — Fluorescence spectroscopy is also a
rapid and efficient method for estimation of population density when the only
particles in suspension are algal cells. The fluorescence spectrophotometer
must be calibrated according to the manufacturer's specifications before each
test and calibrated to zero with growth medium that contains the highest
concentration of toxicant used in the test. If toxicant fluorescence is detected
at the same wavelength as algal fluorescence, this method cannot be used.
The spectrophotometer should be equippped with a recorder for printed direct
readout of results. The printout must be kept in the laboratory notebook.
Wavelengths of excitation and analysis must be determined (see Mitchell
and Kiefer, 1984, for a discussion of absorption and excitation spectra). This
is done by determining fluorescence emission at a specific emission
wavelength while scanning with excitation wavelengths The excitation
wavelength at which emission was greatest is then held constant while the
emission wavelength scale is scanned for greatest emission. The final
excitation and emission wavelengths may be determined by a series of such
steps, using a scanning range more narrow than the previous one.
Wavelengths for M. polymorphus are approximately 443 nm (excitation) and
643 nm (emission). Examine the cultures microscopically for abnormal
cellular morphology If appreciable numbers of abnormal cells are present,
estimate their percentage by differential counts of 100 cells. In order to
determine the IC50 as described below, calculate the average fluorescence
values for untreated control, solvent carrier control, and each treatment group.
Average fluorescence of the carrier controls should be similar to average
fluorescence of the untreated controls. Subtract average fluorescence of Aquil
medium from average fluorescence of the control and treatment cultures for
final fluorescence of the cultures.
CALCULATION OF THE IC50
The IC50 may be calculated by straight-line graphical interpolation (APHA,
1985; Walsh et al., 1987). Plot the toxicant (active ingredient in formulations)
concentration on the y axis and the percentage inhibition on the x axis of
semi-logarithmic graph paper. Plot the concentrations that inhibited growth
just above ana below 50% and draw a line between them. Interpolate the
IC50 as shown in Fig. 3.
27
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CALCULATION OF THE GROWTH RATE
Growth rate (u.) is calculated from the expression
t-t
o
Where N = population density at the end of the test
N0 = population density at the beginning of the test
loginNo = 4.69
t - t0 = length of time of the test (2 days).
Calculate p. from average cell numbers, optical density, or fluorescence
emission of control, solvent carrier control, and each treatment group.
Figure 3. Graphical interpolation of the IC50.
3 -
as
c
10
I
2 -
SC20 = 26 mg/l
TO 15
Waste-Control, Cells
20
25
30
28
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2.15 Fractionation Procedure for Liquid Complex Wastes
If a complex waste inhibits or stimulates algal growth, it is often desirable to
identify its bioactive constituents This may be done by a method described
by Walsh and Garnas (1983), which combines chemical fractionation and
biological testing to identify bioactive organic and inorganic fractions
Bioactive fractions or subfractions may be analyzed chemically to identify
toxic or stimulatory substances. Details of this procedure are given below
2.16 Test for Living and Dead Cells
Algal growth tests do not indicate whether a toxicant simply inhibits
population growth or kills cells directly. It may be desirable to know if a
compound kills algae because, presumably, substances that kill are of more
potential danger than those that inhibit population growth. In this case, algal
populations early in the exponential growth phase are exposed to a toxicant
for 24 h, and differential counts of living and dead cells are performed. Dead
cells are dyed by the mortal stain, Evans blue, in contrast to unstained living
cells. Skeletonema costatum should be used in this test because the cells
are large and easy to identify.
2.17 Bioaccumulation
Algae accumulate toxicants by adsorption to the cell surface and by
absorption into cells. Thus, they constitute the first link in food chain transfer,
whereby toxicants may be transferred to higher trophic levels. Ability of living
algae to accumulate toxicants is estimated by exposure of populations in the
exponential phase of growth to various concentrations in the medium for 24 h,
with subsequent analyses of cell concentrations. It is important that growing
populations, where over 99% of the cells are living, are exposed because
dead cells also adsorb toxicants.
2.18 Documentation and Reporting of Results
When algal tests are done for EPA a notebook that contains detailed
descriptions of methods and quality control procedures used in testing must
be kept in the laboratory at all times. All data from toxicity tests must be
entered in this notebook and these data used for compilation of reports to
EPA.
2.19 References
APHA, 1985 Standard Methods For the Examination of Water and
Wastewater, 16th ed , American Public Health Association, Washington,
DC.
ATCC, 1984. Media Handbook American Type Culture Collection. Rockville,
MD.
Davey, E.W., J.W. Gentile, S.J. Erickson, and P Betzer 1970. Removal of
trace metals from marine culture media Limnol. Oceanogr. 15: 486-
488
Fisher, N.S 1977. On the differential sensitivity of estuanne and open-
ocean diatoms to exotic chemical stress. Am. Nat. 111. 871-895
29
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Fisher, N S , L.B Graham, and E J Carpenter. 1972. Geographic differences
in phytoplankton sensitivity to PCBs. Nature 241: 548-549
Hargraves, P E. and R R L. Guillard 1974. Structural and physiological
observations on some small marine diatoms. Phycologia 13: 163-172.
Hasle, G.R., H.A. von Stosch, and E.E Syvertsen. 1983. Cymatosiraceae, a
new diatom family Bacillaria 6. 9-156
Mitchell, B.G. and D A Kiefer. 1984. Determination of absorption and
fluorescence excitation spectra for phytoplankton. pp. 157-169, in
Lecture Notes on Coastal and Estuarme Studies (O. Holm-Hansen, L.
Bolis and R. Gilles, eds.), Springer-Verlag, New York.
Morel, F M M., J.G. Reuter, D.M. Anderson, and R.R L. Guillard. 1979. Aquil:
a chemically defined phytoplankton culture medium for trace metal
studies J. Phycol. 15 135-141.
Rosowski, J.R. and B C. Parker (eds.) 1982 Selected Papers in Phycology
Phycological Society of America, Inc. Lawrence KS
Starr, R.C 1971. Algal cultures - sources and methods of cultivation, pp.
29-53, in Methods in Enzymology, Vol. 28 (A. San Pietro, ed.),
Academic Press, New York.
Stein, J R (ed.). 1973 Handbook of Phycological Methods: Culture
Methods and Growth Measurements. Cambridge University Press, New
York.
Walsh, G.E. and R.L Garnas. 1983. Determination of bioactivity of chemical
fractions of liquid wastes using freshwater and saltwater algae and
crustaceans. Environ Sci. Technol. 17: 180-182.
Walsh, G E., C.H. Deans, and L.L McLaughlin 1987. Comparison of the EC50
of algal toxicity tests calculated by four methods. Environ. Toxicol.
Chem. 6.767-700.
30
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3. Growth Test with Single Substances
3.1 Preparation of Toxicant
If the toxicant is soluble in growth medium at the highest concentration to be
used in a test, a saturated solution in growth medium should be used as
stock. Have this stock analyzed for purity and concentration.
If the toxicant is of low solubility in growth medium, several days before
the test is to begin prepare a stock solution in nanograde acetone carrier on a
weight to volume basis. Dissolve enough in the acetone so that, when 0.025
ml is added to 51 ml of growth medium, the concentration will approximate
the saturation concentration. Have this stock analyzed for purity and
concentration and store in a sealed flask in an explosion-proof refrigerator.
Express results of chemical analyses as concentration of the toxic substance,
whether it will be tested as a single substance or as the active ingredient in a
formulation.
When toxicant purity and concentration have been confirmed, determine
the highest concentration of toxicant that can be used in the test. This is
done by adding 0.025 ml of the stock to 51 ml of growth medium. If crystals
do not form, this concentration can be used as the highest in the toxicity test.
If crystals form and do not go into solution with swirling of the medium, dilute
the stock slightly and test again for crystal formation. Continue until crystals
do not form. This will be the highest concentration used in the test. This
method cannot be construed to estimate the saturation concentration, but it is
a practical way to determine highest test concentration.
3.2 Toxicant Concentrations
The highest concentration of toxicant to be used in a test is determined as
above, and lower concentrations are percentages of it. Use volumetric flasks
for making dilutions of toxicant stock. In rangefinding tests, the
concentrations are widely separated to estimate the IC50 and establish
chemical concentrations for the definitive test. Use at least five
concentrations, such as 0.01, 0.1, 1, 10, and 100% of the concentration
determined above. All flasks should receive the same volume of toxicant in
carrier, and acetone stock must be diluted so that 0.025 ml is added for each
concentration. Convert percentage to weight of toxicant added and calculate
the concentrations. Toxicant stock and dilutions should be stored in an
explosion-proof refrigerator.
If the substance is toxic to algae in the rangefinding test, use at least five
concentrations of toxicant that encompass the estimated IC50 in the definitive
test. In most cases, each concentration should be at least 60% of the next
31
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higher For example, if, in the rangefinder, there was no inhibition of algal
growth at 10 ppm (parts per million) and total inhibition at 100 ppm, use
concentrations of 13, 22, 36, 60, and 100 ppm in the definitive test. This
sequence, based on 60%, may be unacceptable in some cases, such as
when there is a narrow concentration range between no inhibition and total
inhibition of growth. If, for example, the IC50 is between 5 and 10 ppm, then
the series, 5, 6, 7, 8, 9, and 10 ppm (six concentrations) should be used.
It is clear from the above that there cannot be a single method for
determining concentrations in definitive tests, but that they must be chosen in
relation to algal response. Overall, response must be graded in relation to the
series of concentrations. It is best if at least one concentration inhibits growth
by between 55 and 75%, and another concentration inhibits growth by
between 25 and 45%. These criteria insure that responses to concentrations
used to calculate the IC50 are statistically different from each other, and that
population densities in treated flasks are significantly different from densities
in control flasks
3.3 Test Procedure
Day 1 a. Prepare 2 L of algal growth medium (chelexed or nonchelexed)
as described on page 24, dispense 50 ml into each of 40 125-
ml culture flasks; cap with stainless steel closures or previously
unused foam plugs; label the flasks:
3 control flasks
3 solvent carrier flasks
15 treatment flasks labeled with toxicant concentrations (3
flasks for each concentration)
5 flasks for chemical analyses
1 flask for inoculum culture
1 flask for determination of pH
1 flask to serve as a blank when population density is to be
measured by light or fluorescence spectroscopy
5 flasks that may be needed to replace flasks in which salts
have precipitated
1 flask for dilution of algal inoculum culture
5 extra flasks to be used if needed;
sterilize by heating in an oven at 60 C for 4 h.
b. The following method is recommended for preparation of the
algal growth medium:
1. Dissolve all salts, except MgCla-eHgO in 1.5 L of glass-
distilled or deionized water in a 2-L volumetric flask.
2. After dissolution of salts, add the MgCIa 6H2O
3. After dissolution of MgCIa 6H2O, add 0.5 ml of the PO4",
NOs", and SiOa" nutrients; take to volume and determine
salinity; add 50 ml to culture flasks as above; sterilize.
c. Prepare stock toxicant solution and its dilutions in volumetric
flasks according to the instruction on page 31, wrap masking
tape around the stoppers and flask necks to reduce loss by
evaporation; store in the dark at 4 C in an explosion-proof
refrigerator.
32
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Day 2 a. Examine each flask for presence of precipitated salts by
swirling; if precipitate is present, replace the flask with one
prepared for this purpose.
b. Mix 7.5 ml of metal solution with 7.5 ml of vitamin solution; filter
sterilize as described on page 23; add 0.25 ml of the
metals/vitamins mix to each flask with an Eppendorf pipette
fitted with a sterile tip; flame the mouth of the flask before and
after each addition
c. Prepare an algal inoculum culture by sterile addition of 4-5 ml
of algae from a culture in the logarithmic phase of growth to the
flask prepared on Day 1; place this culture on a shaker at 60
excursions min-1 in the culture chamber dedicated only to
algal tests; incubate for 3 days under conditions identical to
those of the test.
Day 5 a. Measure pH of the medium in the flask prepared on Day 1; if it
is 7.9 - 8.3, continue the test; if not, prepare new medium.
b. The inoculum culture should be in the early exponential growth
phase. Immediately before use, dilute to 500 cells mm-3 by
addition of growth medium prepared for dilution. This is done
by pouring dilution medium into the inoculum culture by sterile
means and estimating population density visually. A sample is
taken with a sterile pipette, added to a hemocytometer, and
cells over the four corner squares and one central square are
counted on both sides of the hemocytometer. At this point, cell
number should be greater than 500 cells mm-3. By careful
addition of dilution medium, cell number can be adjusted
downward to a total of approximately 500 cells mm-3 On both
sides of the hemocytometer.
c. Add 1 ml of diluted algal inoculum culture to all control, carrier
control, and test flasks with an Eppendorf pipette fitted with a
sterile tip; do not add algae to flasks prepared for chemical
analysis; flame the mouth of each flask before and after each
addition; swirl the inoculum culture between each addition to
keep the cells in suspension.
d. Do not add anything more to the three untreated control flasks.
e. With no flame present, add 0.025 ml of earner to media in the
three carrier control flasks; add 0.025 ml of carrier to medium
in one flask to serve as control when a spectrophotometric
method is used to estimate population density; swirl each flask
gently after addition of carrier; do not flame the mouths of the
flasks when adding carrier.
f. Add each concentration of diluted stock toxicant to the proper
set of three test flasks and to each flask prepared for chemical
analysis; swirl each flask gently after addition of toxicant; do not
flame the mouths of the flask when adding toxicant in carrier.
g. Place the flasks, except those prepared for chemical analysis,
on shakers at 60 excursions min-1 in the culture chamber
dedicated only to algal tests.
h. Measure light intensity at the level of the flasks
33
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i. Analyze for the toxicant in the media prepared for chemical
analysis to confirm exposure concentrations.
Day 6 a. Measure light intensity at the level of the flasks
b. Remove all flasks from the shakers and replace at random to
insure equal illumination of cultures.
Day 7 a. Measure light intensity at the level of the flasks.
b. Remove flasks from the culture chamber; estimate population
density by cell counts or absorbance or fluorescence
spectroscopy by methods given in Chapter 2 of this manual.
c. Examine all cultures for presence of abnormal cells; if they are
present, estimate their numbers by the method given on page
26.
d. In rangefinding tests, estimate the IC50 and conduct a definitive
test with five concentrations of toxicant that encompass the
estimated IC50.
e. In definitive tests, calculate the IC50 according to instructions
on page 27; calculate the growth rates of control, carrier
control, and test groups as shown on page 28.
f. Report results on the Reporting Form For Single Substance
Analysis
34
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Reporting Form for Single Substance Analysis
Testing Laboratory
Person in charge
Name of Chemical Compound
Generic
Trademark Soun
Stated Purity (%)
EPA Contract No.
Phone No.
CAS No.
Manufacturer
Date Received
:e
Other Substances in formulation and percentage active ingredient.
Toxicant Stock Solution
Date of preparation
Prepared by:
Method of Chemical analysis
Nominal Concentration Anal}
Purity (%) Anah
Test conditions
Date Begin Date End
pH Salinity
Solvent
/zed Concentration
/zed Purity (%)
Algal Species
Light Intensity:
High Low
Temperature:
High Low Enclose a copy of the Temperature Record
Data recorded in Laboratory Notebook
Number
35
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Test Results
Estimation of Population Density by (Circle):
Cell Counts Optical Density Fluorescence
Range Finder Test Definitive Test
Toxicant Average density
Concentration
1 . Control
2. Carrier
Control
3.
4
5.
6.
7.
8.
Toxicant Average density
Concentration
1 . Control
2. Carrier
Control
3.
4.
5.
6.
7.
8.
IC50 LC50 SC20
36
-------
Growth Rate (y)
Toxicant
Concentration ja
1 . Control
2. Carrier
Control
3.
4.
5.
6.
7.
8.
Cells of Abnormal
Morphology (%)
Toxicant
Concentration %_
1 . Control
2. Carrier
Control
3.
4.
5.
6.
7.
8.
37
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4. Growth Tests with Liquid Complex Wastes
Initial growth tests with liquid complex wastes are conducted on unmodified
whole waste. If the waste inhibits and/or stimulates algal growth, it may be
desirable to fractionate it to determine if effects are caused by organic or
inorganic constituents. The method outlined below was described by Walsh
and Garnas (1983). It describes analysis of whole waste and fractionation
procedures for use with algal tests (Figure 4).
4.1 Collection of Waste
Samples of liquid waste should be collected, shipped, and stored in glass
vessels with Teflon lids or in 20-L polyethylene containers, such as the
Cubitamer® (Cole Farmer Industrial Co., Chicago, IL) or its equivalent. Label
the waste clearly with the name of the site, date, and other data pertinent to
the collection. Give the sample a code number to be used for identification at
all steps in the testing procedure. The waste should be transported to the
laboratory immediately after the sample is taken and, when possible, analysis
should be made immediately upon receipt. Do not store the waste for more
than three days before the first test is done. Samples should be kept at
approximately 4 C during shipment and storage. When received, note color,
odor, and presence of solids, and measure salinity and pH.
4.2 Filtering of Waste
Waste should not be filtered, even though particles may be present, except
when it contains algae. When received, examine it at approximately 100 X
magnification. If algae are present, filter the waste through a 0.45 u.m glass-
fiber filter at no more than 0 5 atm pressure vacuum. Pass 1 L of glass-
distilled or deionized water through the filter before filtering the waste.
4.3 Sterile Techniques
Although the waste is not sterilized, sterile techniques should be used in all
steps of the procedure to avoid addition of bacteria not found at the collection
site.
4.4 Estimation of Population Density
Many liquid complex wastes contain particulates and color that preclude
estimation of population density by electronic particle counters and by light or
fluorescence spectroscopy. If spectroscopic methods are used, blank media
prepared from each waste concentration must be used to calibrate the
38
-------
Figure 4. Scheme for detecting bioactivity of complex waste and its
fractions with algal toxicity tests.
Receive Waste
I
Describe Physical Properties
Salinity, pH
Algal Test
If Bioactive
Organic Fraction Inorganic Fraction
I I
Algal Test Algal Test
If Bioactive If Bioactive
Base/Neutral Acid Particles Anions Cations
I II '
Algal Test Algal Test Algal Test Algal Test Algal Test
instrument to zero before cultures exposed to each concentration are
analyzed. If the waste is highly colored, absorbance spectroscopy may not be
used. If the waste medium fluoresces at the same emission wavelength as the
algae, fluorescence spectroscopy may not be used.
In most cases, population density must be estimated by cell counts using
a hemacytometer.
Examine the cultures microscopically for abnormal cellular morphology. If
appreciable numbers of abnormal cells are present, estimate their percentage
by differential counts of 100 cells. Do this for all tests on waste.
39
-------
Calculate the average cell count, optical density, or fluorescence
emission of untreated control and all waste cultures. Waste may also
stimulate growth, or it may stimulate growth at low concentrations and inhibit
it at higher concentrations. Figure 5 illustrates some types of responses that
can be expected.
Figure 5. Types of responses of unicellular algae to complex liquid
wastes: A inhibition of growth; B stimulation of growth; C stimulation of
growth at low concentrations and in inhibition of growth at high
concentrations.
-------
Control
1
2
3
4
5
% Waste
100
102
105
110
125
160
Waste - Control
-
-
-
10
25
-
Figure 6. Graphical interpolation of the SC20.
5-1
4.
.o
2
I
a
72-h IC50 = 2.9 mg/l
20 40 60 80
Percentage Inhibition
100
41
-------
4.6 Procedure for Effects of Whole Waste
4.6.7 Preparation of Medium
The first test should be done on whole waste, filtered only if algae are
present, without sterilization. After stirring to achieve homogeneity, remove 2
L and add the basic salts (prepared earlier), PCV, NOs", and Si03~. Add
the metals and vitamin mixture as for the test with single compounds, but use
a metal working mix without EDTA.
Store unused waste in the dark at 4 C because it may be needed for
further tests or for fractionation Because waste may change during storage,
subsequent tests should be conducted as soon as possible.
It is best to attempt a definitive test on complex waste immediately in
order to avoid possible effects of storage on toxicity or growth stimulation.
Dilute the waste that contains salts, nutrients, metals (without EDTA), and
vitamins with similar growth medium (without EDTA) prepared with glass-
distilled or deionized water. Make dilutions of 0.001, 0.01, 0.1, 1, 5, 10, 25, 50,
and 75% as follows.
1 2 L of waste = 100%
2 Add 750 ml of 1 to a 1-L volumetric flask and take to volume with
growth medium = 75%
3. Add 500 ml of 1 to a 1-L volumetric flask and take to volume with
growth medium = 50%
4. Add 125 ml of 3. to a 250-ml volumetric flask and take to volume
with growth medium = 25%
5. Add 100 ml of 4. to a 250-ml volumetric flask and take to volume
with growth medium = 10%
6. Add 125 ml of 5. to a 250-ml volumetric flask and take to volume
with growth medium = 5%
7 Add 50 ml of 6. to a 250-ml volumetric flask and take to volume
with growth medium = 1 %
8. Add 25 ml of 7. to a 250-ml volumetric flask and take to volume
with growth medium = 0.1%
9. Add 25 ml of 8. to a 250-ml volumetric flask and take to volume
with growth medium = 0.01%
10. Add 25 ml of 9. to a 250-ml volumetric flask and take to volume
with growth medium = 0.001%
These dilutions were designed to be made in flasks of standard volume with
a minimum amount of diluted waste left after addition to test flasks.
Prepare three test flasks for each waste concentration by dispensing 50
ml into each of three 125 ml Erlenmeyer flasks Also prepare three flasks with
growth medium (without EDTA) as controls and an extra flask of each
concentration to serve as blanks if spectrophotometric methods will be used
for estimation of population density
4.6.2 Procedural Steps
Day 1 a Prepare 3 L of algal growth medium (page 21) in a 3-L
Erlenmeyer flask, deliver 100 ml into each of two 250 ml
42
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Erlenmeyer flasks and 50 ml into each of four 125 ml test
flasks, place a cotton plug in the mouth of each Erlenmeyer
flask and cap the mouths of the test flasks with stainless steel
closures; sterilize all flasks with medium by heating at 60 C for
4 h; store the sterile medium in a draft- and dust-free
cabinet.
b Sterilize 40 125 ml culture flasks capped with stainless steel
closures, a 2-L volumetric flask, and a 50-ml dispensing
pipette by heating at 60 C for 4 h.
Day 2 a. Measure pH of the medium in one of the 50-ml samples
prepared on Day 1; if the pH is between 7.9 and 8.3 continue
with the tests, if not, prepare new medium
b. Add 0 25 ml of filter-sterilized (page 23) metal/vitamin mix to
the flask with, 100 ml medium with an Eppendorf pipette fitted
with a sterile tip; begin an algal inoculum culture in one of them
by adding 2 - 4 ml of an algal culture in the logarithmic phase
of growth with an Eppendorf pipette fitted with a sterile tip; add
0 25 ml of the mixture of metals (without EDTA) and vitamins to
each flask with 50 ml of medium prepared yesterday with an
Eppendorf pipette fitted with a sterile tip, flame the mouth of
each flask before and after each addition.
Note. If algae are present and the waste filtered, absorbance or
fluorescence methods may be used to estimate population density See page
39 for restrictions on use of spectroscopic methods. When such methods are
used, it is necessary to prepare an extra flask with each dilution and undiluted
waste to serve as blanks.
Day 5 a. With the waste at room temperature, add salts and nutrients
(Table 1) to 2 L of waste in the sterile volumetric flask. Dissolve
all salts first except MgCl26H20, then the MgCl2-6H20; add
0.5 ml of the P04", NOa", and SiCV nutrients; then add 10
ml of filter-sterilized metals/vitamin mix (the metals should not
contain EDTA); take to volume with the remaining waste.
b. Dilute the waste to which salts and nutrients were added with
medium prepared on Day 1 according to the method given on
page 42.
c. Dispense 50 ml of undiluted waste and each dilution into each
of three 125-ml test flasks with the sterile dispensing pipette;
this will include 3 flasks for each of the 9 dilutions and 3 flasks
for undiluted waste; label each flask with identifying code,
dilution, and date.
d Measure the pH of undiluted waste and all dilutions with the
portions remaining after addition to the test flasks; if
fluorescence will be used to estimate population density in
filtered medium, determine if the waste fluoresces at excitation
and analysis wavelengths to be used, if it does, fluorescence
may not be used to estimate population density
e Prepare algal inoculum culture by diluting the culture begun on
Day 2 with a portion of the remaining 100 ml of medium
43
-------
prepared on Day 1, dilute to 500 cells mm-3 by the method on
page 33
f Use 3 of the 125-ml culture flasks that contain 50 ml of
medium as untreated controls
g. Add 1 ml of the diluted algal inoculum culture to each flask with
an Eppendorf pipette fitted with a sterile tip, flame the mouth of
each flask before and after each addition
h. Place the flasks at random on shakers at 60 excursions mm-1
in the culture chamber dedicated to algal tests.
i. Measure intensity of light at the level of the cultures.
Day 6 a. Measure intensity of light at the level of the cultures.
b. Remove all flasks from the shakers and replace at random.
Day 7 a. Measure intensity of light at the level of the cultures.
b Remove the algal cultures from the culture chamber and
estimate population density of each by cell counts or by
absorbance or fluorescence spectroscopy; if the waste is not
filtered, count the number of cells as described on page 26; if
the waste is filtered, population density may be estimated by
cell counts or by absorbance or fluorescence spectroscopy.
c If population density is estimated by absorbance, use each
dilution and the undiluted waste (see note on page 43) to zero
the instrument before measuring absorbance of cultures at
comparable dilutions and undiluted waste, or subtract their
absorbances from absorbances of comparable test cultures
d Calculate the IC50, SC20, or both, as described on page 27
and 40, calculate the average growth rate for control and all
waste concentration groups as described on page 28.
e. Report results on the Reporting Form For Complex Waste
Analysis
4.7 Procedures for Effects of Inorganic and Organic Fractions
If whole waste is biologically active, and it is desirable to categorize broadly
its toxic or stimulating components, it may be fractionated into organic and
inorganic components and subfractionated into anion and cation inorganics
and acid and base/neutral extractable organics Also, substances extracted
from particulate matter that is part of the waste may be tested.
Tests on the fractions and subfractions obtained by the methods
described here identify bioactive portions of the waste. They cannot be
construed to express toxicity with great accuracy because of problems
associated with the extraction procedures and because of possible effects of
solvents on algal population growth. Further tests may be required to identify
individual toxic components.
4.7.1 Preparation and Testing of Inorganic and Organic Fractions
On the day that the test on whole waste is placed in the algal growth
chamber, mix the waste well, place 2 L in a glass container, and store in the
dark at approximately 4 C Do not freeze the waste. This portion is to be used
44
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for a test on the whole waste if the first test must be repeated. The remaining
16 L of whole waste will be used for fractionation
Materials
1. 142 mm filter holder
2. 142 mm glass-fiber filter
3. Silicon tubing 1/8" id x 1/4" od
4. 20 L Cubitainer
5. Microflow adjustable gear pump
6. Disposable syringe with Luer Lock® (Becton, Dickenson & Co.,
Rutherford, NJ) tip, 50 ml
7. Amberhte XAD-4 resin
8. Dowex® (Dow Co., Midland, Ml) 1-X8 strong base anion exchange
resin
9. Dowex 50W-X8 strong acid cation exchange resin, 20 - 50 mesh
10 20 mm nylon mesh screen
11 50 Kg scale
XAD-4 Resin Preparation
1 Rinse the resin repeatedly in an Erlenmeyer flask with glass-
distilled or deiomzed water until all salts and fines are removed. Allow
the remaining resin beads to stand in 2N H2SO4 for 30 mm and then
rinse with glass-distilled or deiomzed water.
2. Remove impurities by swirling the resin with technical grade acetone
intermittently for 30 mm, decant, and remove the remaining organic
contaminants by sequential solvent extractions with acetone and
methanol in a Soxhlet extractor for 12 h for each solvent.
3. Transfer the purified resin with distilled methanol into an Erlenmeyer
flask fitted with a ground glass stopper for storage.
Column
The column consists of a 50-ml disposable syringe loosely plugged with
glass wool and filled with 50 ml (wet volume) XAD-4 resin. Use at least
20 bed volumes of glass-distilled or deionized water to displace
methanol from the column. Connect a bored No. 6 Neoprene® stopper
coupled to a 3-cm piece of 8 mm od glass tubing to the top of the
column. A number of columns can be prepared this way and stored in a
refrigerator until needed.
Filter
Position a glass-fiber filter in the filter holder and overlay with 20 pm
nylon mesh to prevent the filter from clogging.
Quality Assurance
In conjunction with the large-volume extraction method, 1-L aliquots of
water sampled before and after resin treatment should be extracted
according to procedures in the "Method 625 Base/Neutrals and Acids"
(Office of the Federal Register, 1986). The fractions obtained from this
procedure, i.e., organic base/neutral and acid can be tested for effects on
algal growth.
Fractionation System
1. Following initial testing of raw effluent, weigh the Cubitainer and its
contents to 0 1 Kg, add a 7 5-cm Teflon stirring bar to the container,
and connect a service faucet to the opening.
45
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2. Connect the Cubitamer to the inlet of the filter assembly with a
minimal length of silicon tubing and connect the column to the filter
exit and to the inlet of the microflow pump with silicon tubing. Direct
the exit of the pump into an empty, tared Cubitamer to receive the
filtered, extracted sample Determine volume of the sample
gravimetrically and store in the dark at approximately 4 C.
3 Be sure to stir the raw effluent in the Cubitainer continuously to
insure homogeneous distribution of solids, and adjust the pump so
that the flow through the apparatus is 500 to 1000 ml tr1. Care must
be taken to avoid overload of the column with subsequent
breakthrough of organics
4 Following extraction of organics, remove the Neoprene stopper and
aspirate the resin by vacuum to remove excess water. Fit the
disposable syringe with an 18 gauge stainless steel needle and elute
with 150 ml nanograde acetone into a rotary evaporator concentrator
flask. Concentrate this organic fraction by vacuum to 25 ml at room
temperature and store, tightly capped, m an explosion-proof
refrigerator.
4.7.2 Procedural Steps
Day 1 Prepare XAD-4 resin and make a resin column as described on
page 45.
Day 2 a Prepare 3 L of algal growth medium m a 3-L flask (page 24),
adding 7.5 ml of each of the PO4", NOa", and S\O^'
solutions; dispense 50 ml into each of four 125-ml culture
flasks and cap with stainless steel closures; dispense 100 ml
into each of two 250-ml Erlenmeyer flasks and plug with
cotton stoppers; plug the 3-L flask with a cotton stopper;
sterilize all flasks with medium by heating at 60 C for 4 h; store
in a draft- and dust-free cabinet.
b. Sterilize 40 125-ml culture flasks capped with stainless steel
closures, two 2-L volumetric flasks, and two 50-ml
dispensing pipettes by heating at 60 C for 4 h.
c. Pass the remaining 16 L of waste through the XAD-4 resin
column as described on page 45. Column effluent will contain
the inorganic fraction and, perhaps, ionized organics. This
fraction will be called the inorganic fraction, even though, in
some instances, it may contain a small amount of organic
material. Store this inorganic fraction in the dark at
approximately 4 C. The organic fraction is the material that
remains on the XAD-4 resin column.
d. Extract the organic fraction from the resin with acetone and
reduce the volume to 25 ml as described above; store in an
explosion-proof refrigerator.
Day 3 a Measure pH of the medium in one of the 50-ml samples
prepared yesterday, if the pH is between 7.9 and 8.3 continue
with the test; if not, prepare new medium.
b. Add 0.5 ml of filter-sterilized (page 23) metal/vitamin mix to
the flasks with 100 ml medium with an Eppendorf pipette fitted
46
-------
with a sterile tip; begin an algal inoculum culture in one of them
by adding 4-5 ml of an algal culture in the logarithmic phase
of growth with an Eppendorf pipette fitted with a sterile tip; add
0.25 ml of the mix of metals (without EDTA) and vitamins to
each of the flasks with 50 ml of medium prepared yesterday
with an Eppendorf pipette fitted with a sterile tip, flame the
mouth of each flask before and after each addition
Preparation and Testing of the Inorganic Fraction
Day 6 a. Prepare algal growth medium with the inorganic column
effluent 6 with salts as described on page 24, add the salts to
approximately 1.5 L of column effluent with continuous stirring
in the sterile 2-L volumetric flask; when the salts have
dissolved, add 0.5 ml of each of the PCV, N03~, and SiOs"
nutrients and 10 ml of the mixture of metals (without EDTA)
and vitamins with an Eppendorf pipette fitted with a sterile tip,
take to volume with the inorganic column effluent and mix
thoroughly; store the remaining column effluent in the dark at
approximately 4 C, if fluorescence spectroscopy is to be used
to estimate algal population density, determine if this sample
fluoresces at the excitation and analysis wavelengths required
for the test algal species; if it does, fluorescence cannot be
used, if absorbance will be used to estimate population density,
determine if color present in the fraction is detectable at the
wavelength used for the algal species; if it is, prepare an extra
flask, as described in c below, for undiluted medium and all
dilutions; use these as blanks for the zero spectrophotometer
settings for comparable test cultures, or subtract their
absorbance values from those of comparable test cultures
b. Add 5 ml of sterile mix of metals (without EDTA) and vitamins
to each liter of dilution medium prepared on Day 2; dilute the
medium prepared from inorganic column effluent according to
instructions on page 42.
c. Dispense 50 ml of undiluted medium and each dilution into 3
sterile test flasks prepared on Day 2 with the sterile dispensing
pipette; this will include 3 flasks for each of the 9 dilutions and
3 flasks for undiluted medium; label each flask with identifying
code, dilution, and date.
d. Measure the pH of undiluted medium prepared from inorganic
column effluent and all dilutions with the portions remaining
after addition to the test flask.
e. Prepare an algal inoculum culture by diluting the culture begun
on Day 3 with a portion of the remaining 100 ml of medium
prepared on Day 2. Dilute to 500 cells mm-3 by the method
on page 33.
f. Use the three flasks with 50 ml medium prepared on Day 2 as
untreated controls
g. Add 1 ml of the diluted algal inoculum culture to each flask with
an Eppendorf pipette fitted with a sterile tip; flame the mouth of
each flask before and after each addition
47
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h. Place the flasks at random on shakers at 60 excursions min~1
in the culture chamber dedicated to algal tests.
i Measure intensity of light at the level of the cultures
Day 7 a Measure intensity of light at the level of the cultures
b. Remove all flasks from the shakers and replace at random.
Day 8 a. Measure intensity of light at the level of the cultures.
b. Remove the algal cultures from the culture chamber and
estimate population density of each by cell counts or by
absorbance or fluorescence spectroscopy.
c If population density is estimated by absorbance, use each
dilution and undiluted medium (see note on page 43) to zero
the instrument before measuring absorbance of cultures at
comparable dilutions and undiluted medium, or subtract their
absorbances from absorbances of comparable test cultures
d Calculate the IC50, SC20, or both, as described on pages 27
and 40; calculate the average growth rate for control and all
waste concentration groups as described on page 28
e Report results on the Reporting Form For Chemical Fraction
Analysis
Preparation and Testing of the Organic Fraction
Day 2 a Prepare 5 L of algal growth medium in a 6-L flask as
described on page 24; add 1.25 ml of each of the PCV,
NOa", and S\0^' solutions, dispense 50 ml into each of four
125-ml culture flasks and cap with stainless steel closures;
dispense 100 ml into each of two 250-ml Erlenmeyer flasks
and plug with cotton stoppers, plug the 3-L flask with a cotton
stopper; sterilize all flasks with medium by heating at 60 C for 4
h; store in a draft- and dust-free cabinet.
b. Sterilize 40 125-ml culture flasks capped with stainless steel
closures, two 2-L volumetric flasks, and a 50-ml dispensing
pipette by heating at 60 C for 4 h
Day 3 a. Measure pH of the medium in one of the 50-ml samples
prepared yesterday; if the pH is between 7.9 and 8 3 continue
with the test; if not, prepare new medium.
b Add 0.5 ml of filter-sterilized metal/vitamin mix (page 23) to
the flasks with 100 ml of medium with an Eppendorf pipette
fitted with a sterile tip; begin an algal inoculum culture in one of
them by adding 4-5 ml of an algal culture in the logarithmic
phase of growth with an Eppendorf pipette fitted with a sterile
tip; add 0.25 of the mixture of metals (without EDTA) and
vitamins to each flask with 50-ml medium prepared on Day 2
with an Eppendorf pipette fitted with a sterile tip; flame the
mouth of each flask before and after each addition
Day 6 a Carefully add 31 ml of the acetone solution of organic
compounds from the XAD-4 resin column to approximately
1.5 L of growth medium prepared on Day 1 in a 2-L
volumetric flask, add 5 ml of the mixture of metals (without
EDTA) and vitamins, bring to volume with growth medium and
mix thoroughly.
48
-------
b. Dilute the medium that contains the organic extract as
described on page 42.
c. Dispense 50 ml of undiluted medium and each dilution to each
of three 125-ml volumetric flasks sterilized on Day 1 with the
sterile dispensing pipette; this will include 3 flasks for each of 9
dilutions and 3 flasks for undiluted medium; label each flask
with identifying code, dilution, and date
d. Measure the pH of undiluted waste and all dilutions with the
portions remaining after addition to the test flasks; if fluores-
cence is used to estimate population density in filtered
medium, determine if the waste fluoresces at excitation and
analysis wavelengths to be used; if it does, fluorescence may
not be used to estimate population density.
e. Prepare an algal inoculum culture by diluting the culture begun
on Day 2 with a portion of the remaining 100 ml of medium
prepared on Day 1, dilute to 500 cells mnv3 by the method on
page 33.
f. Use 3 of the 125-ml culture flasks that contain 50 ml of
medium as untreated controls.
g. Add 1 ml of the diluted algal inoculum culture to each flask with
an Eppendorf pipette fitted with a sterile tip; flame the mouth of
each flask before and after each addition.
h. Place the flasks at random on shakers at 60 excursions min-1
in the culture chamber dedicated to algal tests
i. Measure intensity of light at the level of the cultures.
Day 7 a. Measure intensity of light at the level of the cultures
b. Remove all flasks from the shakers and replace at random.
Day 8 a. Measure intensity of light at the level of the cultures.
b. Remove the algal cultures from the culture chamber and
estimate population density of each by cell counts or by
absorbance or fluorescence spectroscopy.
c. If population density is estimated by absorbance, use each
dilution and the undiluted waste (see note on page 43) to zero
the instrument before measuring absorbance of cultures at
comparable dilutions and undiluted waste, or subtract their
absorbances from absorbances of comparable test cultures
d. Calculate the IC50, SC20, or both, as described on pages 27
and 40; calculate the average growth rate for control and all
fraction concentration groups as described on page 28.
4.8 Procedures for Effects of Inorganic and Organic Subfractions
4.8.1 Inorganic Subfractions
If the inorganic fraction of waste inhibited or stimulated algal growth, it may be
desirable to know whether the effect was due to cations or anions The
following method may be used for this purpose.
49
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4.8.1.1 Preparation and Testing of the Cation Subtraction
Day 1 a. Prepare 5 L of algal growth medium (page 24) in a 6-L
Erlenmeyer flask; deliver 100 ml into each of two 250-ml
Erlenmeyer flasks and 50 ml into each of four 125-ml test
flasks; place a cotton plug in the mouth of each Erlenmeyer
flask and cap the mouths of the test flasks with stainless steel
closures; sterilize all flasks with medium by heating at 60 C for
4 h; store the sterile medium in a draft- and dust-free
cabinet.
b Sterilize 40 125-ml culture flasks capped with stainless steel
closures, five 2-L volumetric flasks, and five 50-ml
dispensing pipettes by heating at 60 C for 4 h.
Day 2 a Measure pH of the medium m one of the 50-ml samples
prepared yesterday, if the pH is between 7.9 and 8.3 continue
with the test, if not, prepare new medium.
b. Add 0 5 ml of filter-sterilized (page 23) metal/vitamin mix to
the flasks with 100 ml medium with an Eppendorf pipette fitted
with a sterile tip, begin an algal inoculum culture m one of them
by adding 4-5 ml of an algal culture in the logarithmic phase
of growth with an Eppendorf pipette fitted with a sterile tip; add
0.25 ml of the mixture of metals (without EDTA) and vitamins to
each flask with 50 ml of medium prepared yesterday with an
Eppendorf pipette fitted with a sterile tip; flame the mouth of
each flask before and after each addition.
Day 3-4 a. Prepare the cation subtraction by batch extraction slowly add 2
N HCI to 2 L of the inorganic fraction until the pH is less than 4;
add 20 g of Dowex 1-X8 strong base anion exchange resin;
stir for 24 h; filter the resin from the sample with the glass fiber
filter assembly; adjust to pH 7 with 2 N NaOH
b. Prepare algal growth medium with the cation subfraction. do
this by adding 1.5 L to a 2-L volumetric flask, dissolving first
all salts except MgCIa 6H2O, then the MgCl2.6H2O, and then
adding 0.5 ml of the P04", NO3", and S\03' nutrients, then
add 10 ml of filter-sterilized metal/vitamin mix (the metals
should not contain EDTA); take to volume with the remaining
waste.
c. Dilute the cation subfraction to which salts and nutrients were
added with medium prepared on Day 1 according to the
method given on page 42.
d. Dispense 50 ml of undiluted cation medium and each dilution
into each of three 125-ml test flasks with the sterile
dispensing pipette, this will include 3 flasks each for the 9
dilutions and 3 flasks for undiluted waste; label each flask with
identifying code, dilution, and date. •
e. Measure the pH of undiluted cation medium and all dilutions
with the portions remaining after addition to the test flasks; if
fluorescence will be used to estimate population density in
filtered medium, determine if the waste fluoresces at the
50
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excitation and analysis wavelengths to be used, if it does,
fluorescence may not be used to estimate population density.
Day 5 a Prepare an algal inoculum culture by diluting the culture begun
on Day 2 with a portion of the remaining 100 ml of medium
prepared on Day 2; dilute to 500 cells mm-3 by the method on
page 33.
b. Use 3 of the 125-ml culture flasks that contain 50 ml of
medium as untreated controls.
c Add 1 ml of the diluted algal inoculum culture to each flask with
an Eppendorf pipette fitted with a sterile tip; flame the mouth of
each flask before and after each addition.
d Place the flasks at random on shakers at 60 excursions mirr1
in the culture chamber dedicated to algal tests
e. Measure intensity of light at the level of the cultures
Day 6 a Measure intensity of light at the level of the cultures.
b Remove all flasks from the shakers and replace at random.
Day 7 a Measure intensity of light at the level of the cultures
b Remove the algal cultures from the culture chamber and
estimate population density of each by cell counts or by
absorbance or fluorescence spectroscopy.
c. If population density is estimated by absorbance, use each
dilution and the undiluted waste (see note on page.43) to zero
the instrument before measuring absorbance of cultures at
comparable dilutions and undiluted waste, or subtract their
absorbances from absorbances of comparable test cultures.
d Calculate the IC50, SC20, or both, as described on pages 27
and 40; calculate the average growth rate for control and all
cation subfraction concentrations as described on page 28.
e. Report results on the Reporting Form For Chemical Fraction
Analysis
4.8.1.2 Preparation and Testing of the Anton Subfraction
Day 1 a Prepare 5 L of algal growth medium (page 24) in a 6-L
Erlenmeyer flask; deliver 100 ml into each of two 250-ml
Erlenmeyer flasks and 50 ml into each of four 125-ml test
flasks, place a cotton plug in the mouth of each Erlenmeyer
flask and cap the mouths of the test flasks with stainless steel
closures; sterilize all flasks with medium by heating at 60 C for
4 h; store the sterile medium in a draft- and dust-free
cabinet.
b. Sterilize 40 125-ml culture flasks capped with stainless steel
closures by heating at 60 C for 4 h.
Day 2 a. Measure pH of the medium in one of the 50-ml samples
prepared yesterday, if the pH is between 7 9 and 8 3 continue
with the test; if not, prepare new medium.
b Add 0.5 ml of filter-sterilized (page 23) metal/vitamin mix to
the flasks with 100 ml medium with an Eppendorf pipette fitted
with a sterile tip; begin an algal inoculum culture in one of them
51
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by adding 4-5 ml of an algal culture in the logarithmic phase
of growth with an Eppendorf pipette fitted with a sterile tip; add
0.25 ml of the mixture of metals (without EDTA) and vitamins to
each flask with 50 ml of medium prepared yesterday with an
Eppendorf pipette fitted with a sterile tip; flame the mouth of
each flask before and after each addition.
Day 3-4 a. Prepare the anion subtraction by batch extraction slowly add 2
N NaOH to 2 L of the inorganic fraction until the pH is greater
than 10; add 20 g of Dowex 50W-X8 strong acid cation
exchange resin (20-50 mesh), stir for 24 h; filter the resin from
the sample with the glass fiber filter assembly, adjust to pH 7
with 2 N HCI.
b. Prepare algal growth medium with the anion subtraction, add
1 5 L to a sterile 2-L volumetric flask, dissolve first all salts
except MgCI26H2O; then dissolve MgCI2-6H20 and add 0.5 ml
of the P04", NO3", and SiOa" nutrients, then add 10 ml of
filter-sterilized metal/vitamin mix (the metals should not
contain EDTA), take to volume with the remaining waste.
c Dilute the anion subtraction to which salts and nutrients were
added with medium prepared on Day 1 according to the
method given on page 42.
d. Dispense 50 ml of undiluted cation medium and each dilution
into each of three 125-ml test flasks with a sterile dispensing
pipette, this will include 3 flasks for each of the 9 dilutions and
3 flasks for undiluted waste; label each flask with identifying
code, dilution, and date
e Measure the pH of undiluted waste and all dilutions with the
portions remaining after addition to the test flasks, if
fluorescence is used to estimate population density in filtered
medium, determine if the waste fluoresces at the excitation and
analysis wavelengths to be used, if it does, fluorescence may
not be used to estimate population density.
Day.5 a. Prepare an algal inoculum culture by diluting the culture begun
on Day 2 with a portion of the remaining 100 ml of medium
prepared on Day 2; dilute to 500 cells mm-3 by the method on
page 33.
b. Use 3 of the 50-ml volume of medium in 125-ml culture
flasks as untreated controls.
c. Add 1 ml of the diluted algal inoculum culture to each flask with
an Eppendorf pipette fitted with a sterile tip; flame the mouth of
each flask before and after each addition.
d Place the flasks at random on shakers at 60 excursions min-1
in the culture chamber dedicated to algal tests
e. Measure intensity of light at the level of the cultures.
Day 6 a Measure intensity of light at the level of the cultures
b Remove all flasks from the shakers and replace at random.
Day 7 a. Measure intensity of light at the level of the cultures
52
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b. Remove the algal cultures from the culture chamber and
estimate population density of each by cell counts or by
absorbance or fluorescence spectroscopy.
c. If population density is estimated by absorbance, use each
dilution and the undiluted waste (see note on page 43) to zero
the instrument before measuring absorbance of cultures at
comparable dilutions and undiluted waste, or subtract their
absorbances from absorbances of comparable test cultures.
d. Calculate the IC50, SC20, or both, as described on pages 27
and 40; calculate the average growth rate for control and all
anion subtraction concentrations as described on page 28.
e. Report results on the Reporting Form For Chemical Fraction
Analysis
4.8.2 Organic Subtractions
If the organic fraction of waste inhibited or stimulated algal growth, it may be
desirable to know whether the effect was due to acid or base/neutral
extractable organics. The extraction method is described in "Method 625
Base/ Neutrals and Acids," published in the 000*6 of Federal Regulations
(Office of the Federal Register, 1986). It is best for the lexicologist who works
with these subtractions to consult that publication for details of the
subfractionation steps and instructions for preparation of the base/neutrals
and acid subtractions of the organic fraction.
4.8.2.1 Preparation and Testing of the Subtractions
Day 1 a. For each subtraction prepare 5 L of algal growth medium (page
24) in a 6-L Erlenmeyer flask; deliver 100 ml into each of two
250-ml Erlenmeyer flasks and 50 ml into each of four 125-ml
test flasks; place a cotton plug in the mouth of each
Erlenmeyer flask and cap the mouth of each test flask with
stainless steel closures; sterilize all flasks with medium by
heating at 60 C for 4 h, store the sterile medium in a draft-
and dust-free cabinet.
b. Sterilize 40 125-ml culture flasks capped with stainless steel
closures by heating at 60 C for 4 h.
Day 2 a. Measure pH of the medium in one of the 50-ml samples
prepared yesterday, if the pH is between 7.9 and 8 3 continue
with the test; if not, prepare new medium.
b Add 0.5 ml of filter-sterilized (page 23) metal/vitamin mix to
the flasks with 100 ml medium with an Eppendorf pipette fitted
with a sterile tip, begin an algal inoculum culture in one of them
by adding 4-5 ml of an algal culture in the logarithmic phase
of growth with an Eppendorf pipette fitted with a sterile tip; add
0 25 ml of the mixture of metals (without EDTA) and vitamins to
each of the flasks with 50 ml of medium prepared yesterday
with an Eppendorf pipette fitted with a sterile tip, flame the
mouth of each flask before and after each addition.
c. Add 3.1 ml of the organic fraction to 2 L of distilled or
deionized water and extract according to instructions given for
53
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separatory funnel extraction in "Method 625-Base/Neutrals
and Acids" of the Code of Federal Regulations. This will yield
two organic subtractions base/neutral and acid in methylene
chloride. Before testing, exchange the methylene chloride with
dimethyl sulfoxide, which is less toxic to algae. Take the
volume to 2 ml and store as instructed in Method 625.
d. The following instructions (Day 6) describe testing with both
extracts. Testing may be done on each at the same or different
times.
Day 6 a. Add 2 ml of the base/neutrals or acids extract to approximately
1.5 L of algal growth medium prepared on Day 1 in a sterile
2-L volumetric flask; add 5 ml of the mix of metals (without
EDTA) and vitamins; bring to volume with growth medium and
mix thoroughly.
b Dilute the medium that contains the organic subtraction as
described on page 42.
c Dispense 50 ml of undiluted medium and each dilution into
each of three 125-ml test flasks with a sterile dispensing
pipette, this will include 3 flasks for each of 9 dilutions and 3
flasks for undiluted waste; label each flask with identifying
code, dilution, and date.
d Measure the pH of undiluted medium and all dilutions with the
portions remaining after addition to the test flask; if
fluorescence is used to estimate population density in filtered
medium, determine if the waste fluoresces at excitation and
analysis wavelengths to be used; if it does, fluorescence may
not be used to estimate population density.
e Prepare an algal inoculum culture by diluting the culture begun
on Day 2 with a portion of the remaining 100 ml of medium
prepared on Day 2; dilute to 500 cells mm-3 by the method on
page 33.
f. Use 3 of the 125-ml culture flasks that contain 50 ml of
medium as untreated controls
g. Add 1 ml of the diluted algal inoculum culture to each flask with
an Eppendorf pipette fitted with a sterile tip; flame the mouth of
each flask before and after each addition.
h Place the flasks at random on shakers at 60 excursions min'1
in the culture chamber dedicated to algal tests.
i. Measure intensity of light at the level of the cultures.
Day 7 a. Measure intensity of light at the level of the cultures.
b Remove all flasks from the shakers and replace at random.
Day 8 a. Measure intensity of light at the level of the cultures
b. Remove the algal cultures from the culture chamber and
estimate population density of each by cell counts or by
absorbance or fluorescence spectroscopy.
c. If population density is estimated by absorbance, use each
dilution and the undiluted waste (see note on page 43) to zero
the instrument before measuring absorbance of cultures at
54
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comparable dilutions and undiluted waste, or subtract their
absorbances from absorbances of comparable test cultures.
d. Calculate the IC50, SC20, or both, as described on pages 27
and 40; calculate the average growth rate for control and all
subtraction concentrations as described on page 28.
e. Report results on the Reporting Form For Chemical Fraction
Analysis.
4.9 Procedures for Extraction and Testing of Particles
The following procedure may be used to determine if organic materials that
can be extracted from waste particulate matter are toxic.
4.9.1 Extraction of Organics
1. Remove the glass fiber filter and nylon screen from the filter holder;
cut into pieces; place in a 150-ml Corex® (Corning Glass Works,
Corning, NY) bottle.
2. Extract the filter pieces 3 times with 50 ml nanograde acetone using
a Branson Sonic Probe for 5 min with each extraction.
3. Centrifuge the extract at 5000 RPM.
4. Concentrate the combined acetone extracts by vacuum at room
temperature to 25 ml.
4.9.2 Testing of Extract
1. The extract contains organics from the particulate matter in 16 L of
waste
2. Test this extract with the method "Preparation and Testing of the
Organic Fraction" page 48.
4.10 References
Office of the Federal Register. 1986. Method 625-base/neutrals and acids.
pp. 442-469. Code of Federal Regulations, 40 CFR 104.1, Revised July
1, 1986, U.S. Government Printing Office, Washington, DC.
Walsh, G.E. and R L. Garnas. 1983. Determination of bioactivity of chemical
fractions of liquid wastes using freshwater and saltwater algae and
crustaceans. Environ. Sci. Technol. 17: 180-182.
55
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Complex Waste Analysis Reporting Form
Testing Laboratory
Person in charge
Origin of Waste
Name of Site
Type of Industry
Sample Collected By
Method of Collection
Method of Transport
Log No.
EPA Contract No.
Phone No.
Address
Date Collected
Date Received
Time of Transport
Color pH Salinity Odor Solids
Other Applicable Characteristics
Test Conditions
Date Begin Date End Algal Species
Light Intensity: High
Low
Temperature:
High Low Enclose copy of Temperature Record
Data recorded in Laboratory Notebook Number
56
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Test Re
Estimation of Population Density
Cell Counts Optical De
First Test
Waste Cone Average density
(%)
1 . Control
2.
3.
4.
5.
6.
7
8.
9.
10.
11.
suits
by (Circle):
snsity Fluorescence
Second Test (If Necessary)
Waste Cone Average density
(%)
i. Control
2.
3.
4.
5.
6.
IC50 LC50 SC20
57
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Chemical Fraction Analysis Reporting Form
Test Conditions
Date Begin
Date End
Algal Species
Light Intensity: High
Low
Temperature:
High Low
Data recorded in Laboratory Notebook Number
Test Results, Fractions
Estimation of Population Density by (Circle):
Cell Counts Optical Density Fluorescence
Fraction
Range Finder Test
Definitive Test
% Original
Cone.
Average
Density
% Original
Cone.
Average
Density
1. Control
2.
3.
5.
1C50
1. Control
2.
LC50
SC20
58
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Growth Rate (p)
Waste
Concentration (%) y
1 . Control
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Cells of Abnormal
Morphology
Waste
Concentration (%) %
1 Control
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
59
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5. Bioaccumulation Test
It may be important to know if algae accumulate a compound. This is
determined by exposure of a population for 24 h, with subsequent chemical
analysis.
1. Dispense 1 L of algal growth medium into six 2.8-L Fernbach flasks,
seal with a foam plug or cotton stopper, and sterilize in an oven at 60
C for 4 h.
2. Add 100 ml of an algal stock culture in the exponential phase of
growth to the flasks and incubate in a growth chamber for five days
Continuous light at 100-120 u.E nv2 sec~1 should be supplied at
20 C. Swirl the flasks twice each day to maintain gaseous exchange
between atmosphere and growth medium
3. On Day 5, add toxicant at the highest concentration that does not kill
algae (see Enumeration of Dead Cells Section) to five flasks, and
return the cultures to the growth chamber for 24 h.
4. After 24 h exposure, centrifuge the cultures at 5000 rpm and
combine all treated algae by resuspension in fresh medium (without
toxicant); add them to a previously weighed centrifuge tube and
centrifuge again.
5. Weigh the tube with algae and subtract the weight of the tube to
determine the weight of algae
6. Analyze for toxicant concentration in algae from the control and
treated flasks and calculate uptake as weight of toxicant per
milligram of wet cells versus weight of toxicant per milliliter of growth
medium. Calculate the concentration factor:
Concentration Factor = toxicant concentration in algae
toxicant concentration in water
7. If concentration of toxicant per milligram of dry algae is desired
a. Dry a filter to constant weight at 110 C.
b. Collect a known wet weight of algal cells suspended in growth
medium on the filter by very gentle vacuum
c. Dry again to constant weight at 110 C and weigh to the nearest
0.1 mg
d Subtract the weight of the filter from the weight of the filter with
algae for dry algal weight.
e. Calculate the concentration factor as described above.
f. Report results on the Bioaccumulation Studies Reporting Form.
60
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NOTE:This method cannot be used if the toxicant is volatile at 110 C There is
a small error in estimation of dry weight of algae by the above method
Some of the salt from the growth medium will be present on the cells and
filter after drying. This is small in relation to algal weight and may be ignored
in this test.
Test Results
Toxicant Concentration in Test
Nominal Analyzed
Bioconcentration
Toxicant Toxicant Cone, in
Concentration Concentration Algae/
Algae Medium Cone, in
Medium
1. Control
2. Treated
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Bioaccumulation Studies Reporting Form
Testing Laboratory
Person in charge
Name of Compound
Chemical
Generic
Trademark
EPA Contract Number
Phone Number
CAS No.
Manufacturer
Date Received
Source
Stated Purity (%)
Other Substances in Formulation and Percentage Active Ingredient
Toxicant Stock Solution
Date of Preparation
Solvent
Prepared by
Method of Chemical Analysis
Nominal Concentration
Purity (%)
Test Conditions
Date Begin
Algal Species pH
Light Intensity: High
Analyzed Concentration
Analyzed
Date End
Salinity
Low
Temperature:
High Low Enclose copy of Temperature Record
Data recorded in Laboratory Notebook Number
62
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6. Enumeration of Living and Dead Cells
The method used here for detection and enumeration of dead algal cells was
described by Crippen and Perrier (1974); Reynolds et al. (1978) and Walsh
(1983) used it to detect effects of toxicants on marine unicellular algae.
Alexander and Wilkinson (1987) showed that toxicants may affect the pattern
of staining
1 Prepare a 1% (w/v) solution of the acidic mono-azo dye, Evans
blue, by dissolving 1 g of the dye in 100 ml of algal growth medium
2. Prepare an algal test with Skeletonema costatum as described for
single substances but do not add toxicant After 48 h, remove the
algal cultures from the growth chamber and add toxicant and solvent
carrier as for the test with single substances. Return the cultures to
the growth chamber and incubate for 24 h.
3. After 24-h exposure, centrifuge the culture gently and resuspend
the cells in a selected volume of uncontammated medium.
Centrifugation should not disrupt cells, time and speed of
centrifugation must be determined by trial and error. Dispense 10 ml
of the suspension into a test tube.
4. Add 0.5 ml of the Evans blue solution to the 10 ml of algal
suspension and wait 30 mm
5. After 30 mm, swirl the suspension to distribute the cells evenly in the
medium and, with a pipette, fill both sides of hemacytometer. Dead
cells will be colored blue.
6. Make a differential count of a total of 100 living and dead cells at
400 x magnification over the grids on each side of the
hemacytometer This can be done by using a hand talley with four
channels, designating one for living cells and one for dead cells on
each side of the hemacytometer
7. Add the living and dead cell counts of each flask and calculate the
percentage of dead cells for the triplicate control, carrier control, and
toxicant concentrations.
8. Calculate the LC50 by straight-line graphical interpolation as shown
in Fig. 3. Plot concentration on the y axis and percentage dead on
the x axis.
9. Report counts of living and dead cells in control, carrier control, and
all toxicant concentrations, giving counts on each of the three
cultures in each of the groups, their averages, and the LC50.
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6.1 References
Alexander, L M and M Wilkinson. 1987. A protocol for the validation of vital
and mortal stains. Bot Mar. 30: 109-113.
Cnppen, R.W and J L Perrier 1974 The use of neutral red and Evans blue
for live-dead determinations of marine plankton. Stain Technol. 49: 97-104.
Reynolds, A E , G.B Mackiernan, and S.D. Van Valkenburg. 1978. Vital and
mortal staining of algae in the presence of chlorine-produced oxidents.
Estuaries 1: 192-196.
Walsh, G E. 1983. Cell death and inhibition of population growth of marine
unicellular algae by pesticides. Aquat. Toxicol. 3' 209-214
.Government Printing Office: 1988 — 548-158/67090
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