ISO/TR 11044:2008
(Main)Water quality — Scientific and technical aspects of batch algae growth inhibition tests
Water quality — Scientific and technical aspects of batch algae growth inhibition tests
ISO/TR 11044:2008 discusses scientific and technical aspects that have been considered in connection with the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and ISO 10253, for marine waters. Previously unpublished results of experiments performed at the Norwegian Institute for Water Research (NIVA) have been included to demonstrate various aspects.
Qualité de l'eau — Aspects scientifiques et techniques des essais d'inhibition de croissance d'un lot d'algues
Kakovost vode - Znanstveni in tehnični vidiki šaržnih preskusov z določevanjem zaviranja rasti alg
To tehnično poročilo obravnava znanstvene in tehnične vidike, ki so bili preučeni v zvezi z razvojem postopkov šaržnih preskusov z določevanjem zaviranja rasti alg, opredeljenih v ISO 8692 za sladko vodo in ISO 10253 za morske vode. Vključeni so še neobjavljeni rezultati eksperimentov, izvedenih na Norveškem inštitutu za vodne raziskave (NIVA), z namenom prikazati različne vidike.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-september-2010
.DNRYRVWYRGH=QDQVWYHQLLQWHKQLþQLYLGLNLãDUåQLKSUHVNXVRY]GRORþHYDQMHP
]DYLUDQMDUDVWLDOJ
Water quality - Scientific and technical aspects of batch algae growth inhibition tests
Qualité de l'eau - Aspects scientifiques et techniques des essais d'inhibition de
croissance d'un lot d'algues
Ta slovenski standard je istoveten z: ISO/TR 11044:2008
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
TECHNICAL ISO/TR
REPORT 11044
First edition
2008-12-01
Water quality — Scientific and technical
aspects of batch algae growth inhibition
tests
Qualité de l'eau — Aspects scientifiques et techniques des essais
d'inhibition de croissance d'un lot d'algues
Reference number
©
ISO 2008
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ii © ISO 2008 – All rights reserved
Contents Page
Foreword. iv
Introduction . v
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 General principles of ISO algal growth inhibition tests.2
5 Test species .4
5.1 General.4
5.2 Pseudokirchneriella subcapitata.6
5.3 Desmodesmus subspicatus .9
5.4 Skeletonema costatum.9
5.5 Phaeodactylum tricornutum.12
6 Test conditions .14
6.1 Growth medium.14
6.2 pH control .16
6.3 Inoculum density .18
6.4 Incubation conditions .21
6.5 Test endpoint .23
Bibliography .25
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
In exceptional circumstances, when a technical committee has collected data of a different kind from that
which is normally published as an International Standard (“state of the art”, for example), it may decide by a
simple majority vote of its participating members to publish a Technical Report. A Technical Report is entirely
informative in nature and does not have to be reviewed until the data it provides are considered to be no
longer valid or useful.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO/TR 11044 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2008 – All rights reserved
Introduction
The growth of microalgae in batch cultures follows a well known pattern, with a lag phase followed by an
exponential growth phase, a phase of declining growth rate, a stationary phase, and ultimately a death phase
(Reference [9]). The characteristics of these phases are dependent on the environmental conditions including
the chemical composition of the growth medium, which provides the basis for using batch cultures of algae as
bioassays to investigate growth stimulating or inhibiting properties of constituents of the growth medium.
The first systematic application of microalgae bioassays for which standard protocols were developed was for
assessment of nutrient status and identification of growth limiting nutrients. Skulberg (Reference [50])
developed a bioassay with the green alga Selenastrum capricornutum Printz, which was used to assess
fertilizing influences of pollution in inland waters. The nutrient bioassay with S. capricornutum was further
developed and standardised in Reference [55]. The strain of S. capricornutum used as test organism in the
nutrient bioassays was originally isolated from the river Nitelva in southeast Norway in 1959. It has become
the most commonly used test algae for bioassays and is available from most major culture collections. Due to
taxonomic revisions, it was first renamed Raphidocelis subcapitata and later Pseudokirchneriella subcapitata
(Korshikov) Hindak (Reference [20]).
It was early recognized that bioassays of microalgae could be used to study the growth-inhibiting effects of
toxic chemicals and waste waters, and a modification of the algal assay procedure for toxicity studies was
made in Reference [43]. However, based on compilations of early algae toxicity test data some authors
claimed that the sensitivity of algae generally was low (Reference [26]). The environmental relevance of
results of the tests was also questioned because of the significant interspecies variation in response and lack
of field-validation of results of algal toxicity tests (Reference [28]). On the other hand, microalgae are generally
the most important primary producers in aquatic ecosystems. Excluding the assessment of toxicity to this
group of organisms in risk assessment and environmental management cannot be justified. Development and
standardisation of methods have therefore been undertaken to increase the reproducibility and relevance of
toxicity tests with microalgae. Standardised growth inhibition tests with algae are now a cornerstone in the
environmental management and risk assessment of chemicals. Recent reviews (e.g. Reference [57]) show
that they are often the most sensitive of the “base-set” tests which include also acute toxicity tests with fish
and Daphnia.
In addition to several national organisations, the Organisation for Economic Co-operation and Development
(OECD) and the International Organization for Standardization (ISO) took on the work of developing
guidelines and standards for growth inhibition with microalgae in the late 1970s. The OECD guidelines aim to
test chemical substances, while ISO documents cover tests for composite water samples, such as waste
water and elutriates. However, harmonisation of the procedures was an objective as the two series of
documents were developed in parallel by the two organisations. The development of the freshwater test was
initiated by ISO in 1978. Three ring tests were organised between 1980 and 1982 and included in
ISO 8692:1989, revised as ISO 8692:2004. The first draft of a marine algae inhibition test was produced in
1982, but the first ISO/DIS was not published until 1991, when the method had been ring tested.
ISO 10253:1995 was revised as ISO 10253:2006. In addition to these two standards, ISO 14442:1999,
guidelines for algal growth inhibition tests with poorly soluble matter, volatile compounds, metals and waste
water, was revised as ISO 14442:2006. In this Technical Report, the general principles of the batch culture
growth inhibition tests, and how some critical methological aspects have been addressed in the International
Standards for algal growth inhibition tests, are presented.
TECHNICAL REPORT ISO/TR 11044:2008(E)
Water quality — Scientific and technical aspects of batch algae
growth inhibition tests
1 Scope
This Technical Report discusses scientific and technical aspects that have been considered in connection with
the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and
ISO 10253, for marine waters.
Previously unpublished results of experiments performed at the Norwegian Institute for Water Research
(NIVA) have been included to demonstrate various aspects.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 8692:2004, Water quality — Freshwater algal growth inhibition test with unicellular green algae
ISO 10253:2006, Water quality — Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum
ISO 14442, Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile
compounds, metals and waste water
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
effective concentration
EC
x
concentration of test sample which results in a reduction of x % in the specific growth rate relative to the
controls
[ISO 8692]
NOTE Unless otherwise stated, the form EC is used in this Technical Report to mean E C where “r” denotes “rate”.
x r x
Effective concentrations based on area under the growth curve can be derived, and these are designated E C , where “b”
b x
denotes “biomass” (see 6.5 for further details).
3.2
specific growth rate
µ
proportional rate of increase in cell density per unit of time:
1dn
µ =
ntd
where
n is the cell density, expressed in cells per millilitre;
t is the time, expressed in days.
NOTE 1 Specific growth rate is expressed in reciprocal days.
NOTE 2 Adapted from ISO 8692.
4 General principles of ISO algal growth inhibition tests
The algae growth inhibition test methods specified in ISO 8692 and ISO 10253 are based on batch cultures
which are inoculated with algae from an exponentially growing inoculum culture and incubated under
continuous illumination. The growth medium, inoculum biomass density, temperature, and illuminance, have
been selected to allow an exponential increase in the algal biomass density during the 72 h incubation period
for the recommended test species.
The experimental design of the tests includes a series of five or more concentrations of the test material in
growth medium prepared in triplicate, and six control replicates without test material. After inoculation with test
algae, the solutions are incubated in transparent, inert containers under continuous illumination and constant
temperature. The cultures should be agitated in order to obtain a homogenous suspension of the algae and to
stimulate gas exchange with the atmosphere. The biomass density in the cultures is measured by direct or
indirect methods at 24 h intervals until termination of the test after 72 h.
An example of a growth inhibition test with Pseudokirchneriella subcapitata is shown in Figure 1. The
substance tested was potassium dichromate. The growth curves show close adherence to exponential growth
in the cultures, and decreasing growth rates with increasing concentration of the test substance. Average
specific growth rates may be calculated as the logarithmic increase in cell density from start to 72 h. Figure 2
shows the concentration/response plot for the endpoint growth rate. A curve has been fitted to the
1)
observations by non-linear regression using a log-logistic model (REGTOX) . Concentrations causing 10 %
and 50 % reduction of the growth rate (EC and EC respectively) have been calculated from the regression
10 50
equation.
1) Available (2008-11-14) at http://eric.vindimian.9online.fr/
2 © ISO 2008 – All rights reserved
Key
1 ρ(K Cr O ) = 0 (control) 5 ρ(K Cr O ) = 1 mg/l
2 2 7 2 2 7
2 ρ(K Cr O ) = 0,25 mg/l 6 ρ(K Cr O ) = 1,6 mg/l
2 2 7 2 2 7
3 ρ(K Cr O ) = 0,4 mg/l n cell density, 10 cells/ml
2 2 7
4 ρ(K Cr O ) = 0,63 mg/l t time, h
2 2 7
Figure 1 — Growth curves (mean values of replicates) for cultures of P. subcapitata
at different mass concentrations of K Cr O
2 2 7
Key
µ specific growth rate as a percentage of control
EC effective concentration at 10 % inhibition
EC effective concentration at 50 % inhibition
ρ(K Cr O ) potassium dichromate mass concentration, mg/l
2 2 7
Figure 2 — Mass concentration/response plot showing the effect of K Cr O
2 2 7
on the growth rate of P. subcapitata
5 Test species
5.1 General
Microalgae constitute a phylogenetically diverse group of organisms, including the procaryotic cyanobacteria
and several phyla of eucaryotic algae. It is therefore not surprising that the sensitivity among different species
of microalgae to various toxic substances is highly variable. Some studies have shown that such interspecies
variation in sensitivity may amount to three to four orders of magnitude (References [2], [24], [54]). This
variation in sensitivity must, of course, be acknowledged when intrepreting data on algal toxicity in a risk
assessment context and the use of a battery of species has been proposed to account for the variation
(References [8], [21], [33], [53]).
ISO 8692 specifies two green algae — P. subcapitata and Desmodesmus subspicatus (previously known as
Scenedesmus subspicatus) — as test species in freshwater. ISO 10253 specifies two marine diatoms,
Skeletonema costatum and Phaeodactylum tricornutum for the marine algae growth inhibition test. A search
for data entries on toxicity of chemicals to the algal species included in the ISO and OECD test methods in the
US EPA database ECOTOX showed a total of approximately 5 000 data entries of which 42 % are from tests
with P. subcapitata, which confirms the position of this strain as a reference alga in bioassays (see Figure 3).
Among the marine species, S. costatum appears to be the one most frequently used.
4 © ISO 2008 – All rights reserved
Key
1 P. subcapitata 5 Navicula pelliculosa
2 Chlorella vulgaris 6 S. costatum
3 D. subspicatus 7 P. tricornutum
4 Anabaena flos-aquae n number of entries
e
a
Freshwater algae.
b
Marine algae.
Figure 3 — Number of data entries on toxicity to algae in the US EPA database ECOTOX
Some characteristics of the ISO 8692 and ISO 10253 test algae are presented in Table 1. The data were
obtained from batch cultures in ISO 8692 (freshwater) and ISO 10253 (sea water) media. The cultures were
2 2)
incubated at 21 °C and continuous illuminance of 80 µmol/m s and analysed in the late exponential phase.
3)
The cell density and mean cell volume were measured using a Coulter Multisizer M3 equipped with a
100 µm orifice tube. The dry mass was measured after collection of the algae on a glass fibre filter which was
dried at 104 °C until constant mass. For the marine species the mass of salts in the water adsorbed in the
filters was corrected for. It should be noted that “cell” in this context refers to particles identified by the particle
counter. For species forming aggregates as e.g. D. subspicatus and S. costatum, the true cell volume and
mass may be less than indicated in Table 1.
2) Both ISO 8692 and ISO 10253 use the term “light intensity” rather than “illuminance”. The photosynthetically available
radiance (PAR) is defined as the total irradiance in the wavelength range 400 nm to 700 nm. Both ISO 8692 and
ISO 10253 indicate in a note that for light-measuring instruments calibrated in the photometric unit, lux, an equivalent
range of 6 000 Ix to 10 000 Ix is acceptable for testing.
3) Example of a suitable product available commercially. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of this product.
Table 1 — Example of size and mass of cells of different ISO test algae
grown in freshwater and marine growth media
Mean cell volume Mean cell dry mass
Species Strain
µm mg
−8
P. subcapitata NIVA/CHL 1 ≡ CCAP 278/4 72 3,0 × 10
−8
D. subspicatus NIVA/CH 55 ≡ SAG.86.81 139
5,3 × 10
−8
S. costatum NIVA/BAC 1 115
4,6 × 10
−8
P. tricornutum
NIVA/BAC 2 56 1,9 × 10
5.2 Pseudokirchneriella subcapitata
P. subcapitata is the most used test alga in growth inhibition tests and is recommended as test species in
several national standards in addition to the international ISO and OECD test protocols. All cultures of this
species maintained in the major culture collections (e.g. CCAP 278/4, ATCC 22662, 61.81 SAG, UTEX 1648)
stem from a clone culture isolated from a Norwegian river in 1959 (Reference [50]). This is a great advantage
from the point of view of reproducibility of test results which is an important aspect of standardisation. The
appearance of P. subcapitata in culture is shown in Figure 4.
a) P. subcapitata
b) D. subspicatus
c) S. costatum
d) P. tricornutum
Figure 4 — Light microscope photographs of cultures of test algae specified
in ISO 8692 and ISO 10253
6 © ISO 2008 – All rights reserved
The cells are solitary and easily counted with an electronic particle counter. Up to eight autospores form within
the cells and are released when the daughter cells are mature. As a result, a non-synchronous culture
contains a mixture of cells of various sizes from small, recently released cells to large cells with visible
autospores. An example of the size distribution of such a culture analysed with a Coulter Multisizer is shown in
Figure 5.
Sometimes, partial synchronisation occurs in toxicity tests with P. subcapitata. In such a case, a size
distribution with two peaks may be observed.
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n V
differential cell number differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 5 — Size distributions of a culture of P. subcapitata
in the exponential growth phase in ISO 8692 growth medium
The requirement for growth of P. subcapitata in the control cultures in ISO 8692 is that the specific growth rate
−1
shall be at least 1,4 d . This corresponds to an increase in cell density of a factor of 67 in 72 h. Normally the
growth rate is well above this requirement under the conditions specified in ISO 8692. Results of an
experiment where cultures of P. subcapitata were incubated on a light/temperature gradient are shown in
Figure 6.
Key Key
2 2
1 14,5 °C 5 29,1 °C 1 30 µmol/m s 5 90 µmol/m s
2 2 2
2 19,6 °C E illuminance, µmol/m s 2 50 µmol/m s 6 100 µmol/m s
−1 2
3 22,7 °C µ specific growth rate, d 3 70 µmol/m s T temperature, °C
2 −1
4 25,6 °C 4 80 µmol/m s µ specific growth rate, d
NOTE The range of illuminance specified in NOTE The range of temperature specified in ISO 8692
ISO 8692 is indicated by the frame inside the figure. is indicated by the frame inside the figure.
a) plotted against illuminance b) plotted against temperature
Figure 6 — Effects on the growth rate of P. subcapitata in ISO 8692 growth medium
In this experiment, the cell density was measured twice every day for 5 d, and the growth rate calculated by
regression analysis of cell density against time for the exponential part of the growth curve. The results
indicate that the maximum growth rate occurs at a temperature of approximately 32 °C. In the temperature
range specified in ISO 8692 (21 °C to 25 °C) a linear increase of growth rate with temperature was observed.
2 −1
At an illuminance of 90 µmol/m s, the growth rate increased by 0,16 d for each degree celsius between
2 −1 −1
17 °C and 27 °C (R = 0,995 1). This means that the growth rate increased from 1,38 d at 21 °C to 2,02 d
at 25 °C.
The effect of illuminance was highly temperature dependent. At temperatures below 20 °C, the growth rate
2 2
was almost unaffected by illuminance in the range 30 µmol/m s to 100 µmol/m s. At higher temperatures,
−1
the growth rate increased with illuminance, but even at 22,7 °C, the growth rate increased only 0,2 d
2 2
between 60 µmol/m s and 100 µmol/m s. The growth response at higher temperatures indicates that growth
is saturated at about 90 µmol/m s. The results are in agreement with Reference [35], which reports growth
−1 2 −1 2
rates of approximately 1,31 d at 50 µmol/m s and 1,51 d at 100 µmol/m s at a temperature of 20,8 °C.
8 © ISO 2008 – All rights reserved
5.3 Desmodesmus subspicatus
Different strains of D. subspicatus can be obtained from the major culture collections. The strain specified in
ISO 8692 (SAG 86.81 ≡ CCAP 276/22 ≡ UTEX 2594) was originally isolated by Brinkmann from an aquarium
in Berlin in 1953. According to Reference [17], this strain grows mainly as single cells in culture. It may,
however, also occur in colonies (coenobia) of four cells to eight cells arranged in a row or as loose aggregates
without systematic organisation [see Figure 4 b)]. Cell length and width reported in Reference [17] are 3,5 µm
to 5,0 µm and 4,0 µm to 6,0 µm, respectively. The size distribution of a culture in ISO 8692 medium is shown
in Figure 7.
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n differential cell number V differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 7 — Size distributions of a culture of D. subspicatus in the exponential growth phase
in ISO 8692 growth medium
The size distribution plots indicate that the single cells with a spherical diameter of approximately 4 µm to
7 µm dominate. The long tail in the distributions with spherical diameters up to at least 15 µm, which is
especially pronounced in the differential volume plot, is probably caused by the presence of aggregates.
5.4 Skeletonema costatum
Several strains under the name of S. costatum are available in the major culture collections. In ISO 10253,
two strains are specified: CCAP 1077/1C (origin: North Sea, post-1970) and NIVA BAC 1 (origin: Oslo Fjord,
1962). In the CCAP strain catalog, two other strains (CCAP 1077/3 and 1077/5) are listed as strains used for
ecotoxicity testing, while this is not the case for CCAP 1077/1C. Probably several different strains are used as
test organisms. The issue is further complicated by recent taxonomic revisions which have revealed that
S. costatum is a “species complex” rather than a true species, and several previous “S. costatum” strains have
been renamed as S. grethae (e.g. CCAP 1077/3 and 1077/4) or S. pseudocostatum (CCAP 1077/7) (see
Reference [49]). Apparently CCAP 1077/1C has not yet been examined for taxonomic revision. The stain
NIVA BAC 1 has recently been examined and allocated to S. pseudocostatum.
The size distribution of the strain NIVA BAC 1 grown in ISO 10253 medium is shown in Figure 8. The
distribution shows a fairly narrow peak around 6 µm, indicating that the culture is predominantly single celled
or that the particle counter is able to distinguish each cell even if they are connected in chains [see
Figure 4 c)].
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n differential cell number V differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 8 — Size distributions of a culture of S. costatum strain NIVA BAC 1 (S. pseudocostatum)
in the exponential growth phase in ISO 10253 growth medium based on natural sea water
During the development of ISO 10253, a comparison of different S. costatum strains was performed
(Reference [13]). The strains included were ISTPM P4, NIVA BAC 1, CCAP 1077/1C, CCAP 1077/3, and
CCAP 1077/5. The study showed differences in cell size and chain length between the strains and suggested
that these differences were correlated to differences in sensitivity to the reference substances potassium
dichromate and 3,5-dichlorophenol (DCP). The results are summarized in Table 2.
Table 2 — Results of tests of reference substances with different strains of S. costatum
Potassium dichromate 3,5-Dichlorophenol
Control growth Mean particle size in
rate control
EC EC
Strain
50 50
−1
mg/l mg/l µm /cell
d
ISTPM P4 6,6 1,4 2,64 to 3,36 563
NIVA BAC 1 2,6 0,95 1,68 to 2,11 120
CCAP 1077/1C 3,9 1,2 1,65 to 1,94 169
CCAP 1077/3 7,4 1,4 2,64 to 2,40 596
CCAP 1077/5 5,7 1,3 2,25 to 2,40 454
In future revisions of ISO 10253, the implications of the recent taxonomic revision of the “Skeletonema
costatum complex” should be considered. Preferably only one strain of S. costatum should be recommended
as test alga.
10 © ISO 2008 – All rights reserved
The effect of temperature on the growth rate of strain BAC 1 in ISO 10253 growth medium based on natural
sea water is shown in Figure 9. The specific growth rate was calculated from the increase in biomass density
(measured as total cell volume with a Coulter Multisizer) in batch cultures after 70 h incubation at
80 µmol/m s. The results showed an almost linear increase in growth rate with temperature in the range
−1 2
13 °C to 21 °C. Linear regression of the data show a slope of 0,141 d per degree celsius (R = 0,996 2). The
optimum temperature for this strain appears to be around 24 °C and growth was completely inhibited at
32,5 °C. It should be noted that the growth rates observed in this experiment are somewhat lower than what is
normally found in control cultures in toxicity tests with the same strain under similar conditions. The specific
growth rates obtained in control cultures incubated at approximately 75 µmol/m s and 21 °C are usually
−1
around 2,0 d . The low calculated growth rates in the experiment are probably due to the fact that the growth
rate was not exponential throughout the incubation period.
Key
T temperature, °C
−1
µ specific growth rate, d
Figure 9 — Effect of temperature of average growth rate (0 h to 70 h) of S. costatum (NIVA BAC 1)
incubated in ISO 10253 natural sea water medium at 80 µmol/m s
Because of the high growth rate of most S. costatum strains (see Table 2), exponential growth is often not
maintained for 72 h under the specified test conditions, even if the inoculum density is held at 5 × 10 cells/ml.
This may have the effect that partly inhibited cultures catch up with the control cultures during the last day of
the test as shown in Figure 10 a). In this example, the significant growth inhibition that was observed in 1 mg/l
of 3,5-DCP after 48 h was no longer seen after 72 h. Deviation from exponential growth of S. costatum was
noted also in the ISO and PARCOM ringtests (References [1], [15]). It is obvious that it is more appropriate in
this case to base the calculations of growth rates on the first 48 h of the test when the growth in the control
cultures is still exponential. In ISO 10253:2006, such a recommendation was included.
Key Key
1 ρ(3,5-DCP) = 0 (control) 5 ρ(3,5-DCP) = 2,5 mg/l 1 ρ(3,5-DCP) = 0 (control) 5 ρ(3,5-DCP) = 4 mg/l
2 ρ(3,5-DCP) = 0,63 mg/l 6 ρ(3,5-DCP) = 4 mg/l 2 ρ(3,5-DCP) = 1 mg/l
3 3
3 ρ(3,5-DCP) = 1 mg/l n cell density, 10 cells/ml 3 ρ(3,5-DCP) = 1,6 mg/l n cell density, 10 cells/ml
4 ρ(3,5-DCP) = 1,6 mg/l t time, h 4 ρ(3,5-DCP) = 2,5 mg/l t time, h
a) S. costatum (NIVA BAC 1) b) P. tricornutum (NIVA BAC 2)
Figure 10 — Growth curves obtained from 3,5-dichlorophenol toxicity tests
5.5 Phaeodactylum tricornutum
The strains of P. tricornutum specified in ISO 10253 are CCAP 1052/1A (origin: off Plymouth, UK) and
NIVA BAC 2. The origin of BAC 2 is not known. It was obtained from the University of Oslo and it may be
identical to CCAP 1052/1A. Size distributions of a culture of the strain BAC 2, grown in ISO 10253 medium
based on natural sea water, are shown in Figure 11. P. tricornutum appears as single cells in culture and the
variation in cell size is usually small. Typical growth rates of this strain in ISO natural sea water medium are
−1 2
around 1,6 d at 21 °C and 75 µmol/m s.
From a technical point of view, P. tricornutum is an excellent test organism (low variation in cell size, does not
form aggregates, intermediate growth rate). In contrast to S. costatum, exponential growth is usually
maintained for 72 h under the test conditions specified in ISO 10253 [see Figure 10 b)]. In spite of this,
P. tricornutum has been used less frequently than S. costatum in marine algae toxicity tests (Figure 3). The
main reason for this is probably that S. costatum is considered as a more relevant species from an ecological
point of view. Furthermore, the requirement to use S. costatum as a test organism in the PARCOM test
programme for offshore chemicals has contributed to its popularity. Nevertheless, P. tricornutum was included
in a ring test organised by PARCOM in 1991. In comparison to S. costatum, P. tricornutum was less sensitive
to the reference substance 3,5-DCP (which is in agreement with the result of the ISO ring test) but more
sensitive to two offshore chemicals (Reference [1]).
P. tricornutum inhabits brackish waters and has a wider salinity tolerance than S. costatum. It is therefore a
suitable species for tests in brackish waters or for testing toxic effects of “fresh” waste waters in sea water. By
selection of different species specified in ISO 8692 and ISO 10253, toxicity tests may be performed at all
salinities from freshwater to sea water. Figure 12 shows the effect of salinity on the growth rate of
P. subcapitata, P. tricornutum (NIVA BAC 2), and S. costatum (NIVA BAC 1) in ISO media prepared with
different proportions of distilled water and natural sea water.
12 © ISO 2008 – All rights reserved
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n differential cell number V differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 11 — Size distributions of a culture of P. tricornutum strain NIVA BAC 2 in the exponential
growth phase in ISO 10253 growth medium based on natural sea water
Key
−1
1 P. subcapitata µ specific growth rate, d
2 P. tricornutum ρ(NaCl) salinity (sodium chloride mass concentration), g/l
3 S. costatum
Figure 12 — Growth of P. subcapitata, P. tricornutum (NIVA BAC 2),
and S. costatum (NIVA BAC 1) at different salinities
6 Test conditions
6.1 Growth medium
The composition of the growth medium may affect the toxic response of the test algae, either because of
chemical interactions that affect the biological availability of the test substance or because of its effects on the
physiological status of the algae. Influence of the growth medium composition on the expression of toxicity in
algal tests has in particular been demonstrated in tests of toxic metals, where pH, hardness and presence of
chelating agents are important factors (Reference [22]). Of course, the same factors affect the toxicity to algae
in their natural environments. A standardised growth medium can therefore not be representative to all natural
environments, a fact that must be recognised when interpreting toxicity data from tests with algae as well as
for other aquatic organisms.
Test media for the ISO growth inhibition tests have been designed to allow unrestricted exponential growth of
the recommended test algae under the specified culturing conditions, avoiding as much as possible excessive
concentrations of ingredients that may cause chemical interference with various test substances. For the
freshwater test (ISO 8692) a specific growth medium was developed after experience from early ring tests
indicated that differences in composition of the medium affected the test results. It was also concluded that a
high buffering capacity was required in order to reduce pH variations which were identified as one of the main
reasons for deviations in results between different laboratories (Reference [13]). In the third ISO ring test, a
−
medium buffered with 150 mg/l NaHCO and NO nitrogen source were used. Still, a substantial pH increase
was observed after 96 h incubation (Reference [14]). Later it was decided to restrict the exposure to 72 h, and
modifications of the medium were made, including lowering the concentration of NaHCO to 50 mg/l and
− +
replacing K HPO with KH PO . NO was replaced with NH as a nitrogen source in order to counteract pH
2 4 2 4 3 4
increase that tended to occur towards the end of the test when the CO demand from algal photosynthesis
exceeded the mass transport of CO across the water/gas interface.
Chelating agents are required to avoid precipitation of iron in the growth medium. However, chelators may
reduce the availability and effect of toxic metals. EDTA (ethylenediaminetetraacetate) is the most commonly
used chelator in artificial algal growth media. In ISO 8692:1989, the medium contained 80 µg/l FeCl ·2H O
3 2
and 100 µg/l of Na EDTA·2H O. Later, the iron content was reduced to 64 µg/l of FeCl ·2H O in order to
2 2 3 2
obtain equimolar concentrations of EDTA and complexing trace metals. Still the medium is not optimal for
testing metal toxicity. Different modifications of the medium have been used to overcome this shortage. In
Reference [18], 32 µg/l humic acid replaced EDTA in order to create a medium with metal complexing
properties more similar to natural surface waters. Some reported tests (e.g. Reference [31]) employed
synthetic growth media without organic chelators. Experience has shown, however, that the growth of algae is
highly variable in such media. Iron is required as a trace element in the medium, and the concentration
required to support the production of biomass in a 72 h test is above the solubility of iron(III) hydroxide. The
effect of excluding EDTA in the medium is demonstrated in Figure 13, which shows the growth performance of
P. subcapitata in the ISO medium with and without EDTA. The two media were inoculated with 5 × 10
cells/ml of P. subcapitata grown in medium with EDTA. The average specific growth rate in six cultures with
−1
EDTA was 1,68 d with a coefficient of variation of 2,9 %. In nine cultures without EDTA, the average growth
−1
rate was 1,15 d with a coefficient of variation of 21 %.
Studies performed at the Danish Technical University demonstrated that the concentration of iron and EDTA
could be reduced without affecting the growth performance of P. subcapitata and based on this finding, a
recommendation for a modified medium for testing of metals was included in ISO 14442, a guidance
document for tests with poorly soluble materials, volatile compounds, metals and waste water. The proposal is
to use 20 µg/l (0,074 µmol/l) of FeCl ·2H O and 22,6 µg (0,083 µmol/l) Na EDTA·2H O in the medium and to
3 2 2 2
reduce the test duration from 3 d to 2 d.
14 © ISO 2008 – All rights reserved
Key
1 with EDTA
2 without EDTA
n
cell density, 10 cells/ml
t
time, h
Figure 13 — Growth of P. subcapitata in ISO 8692 growth medium with EDTA (0,27 µmol/l)
and without EDTA (mean values and standard deviation)
The ISO 10253 marine test can be performed in synthetic or natural seawater of (30 ± 5) g/kg salinity. The sea
water is spiked with nutrients in order to support exponential growth of the test algae for 72 h. The nutrient
[58]
medium has been adopted from ASTM E 1218 . Because of the high content of anions in the sea water, a
higher concentration of EDTA than in the freshwater medium is required to avoid precipitation of iron. During
the development of the ISO 10253 test, some work was done to investigate the possibility of reducing the
content of EDTA in the medium in order to make it more suitable for testing metals. Experiments at NIVA
performed in 1991 showed that the BAC 1 strain grew as well in a natural sea water medium with iron and
EDTA reduced to the levels used in the freshwater medium (ISO 8692) provided that the zinc concentration
was also reduced from 150 µg/l to 15 µg/l. Reduced concentrations of iron and EDTA were also used by
several laboratories that participated in a ring test organised by PARCOM in 1991 (Reference [1]). Some
laboratories reported, however, that S. costatum would not grow as well in the modified medium as in the
original ISO 10253 medium. This may be due to the differences in the composition of the natural sea water
used by different laboratories and the fact that different strains of S. costatum may have different nutrient
requirements. It was found that any modifications of the ISO 10253 medium would need to be based on
extensive trials in many laboratories and the original formulation with 149 µg/l iron and 15 mg Na EDTA·2H O
2 2
has been retained.
Since the growth inhibition tests have been designed to allow exponential growth in a batch culture, the algae
will not experience nutrient limitation. This is different from the natural habitats of most microalgae, where the
growth is often limited by the supply of a limiting nutrient. It has been argued that the stress from nutrient
limitation may increase the sensitivity of algae to toxic substances and some studies indicate that this may be
the case for the effects of metals on P. subcapitata (References [3], [30]). Other studies have also shown that
effects on nutrient uptake by algae may occur at lower concentrations of toxicants than those affecting growth
(References [38], [46]), and such effects may have important consequences for the competitive ability of algae
in a nutrient-limited environment. However, it may also be argued that the potential for a toxicant to cause a
significant effect on the growth of an alga is higher when the growth rate is high than when it is reduced as a
consequence of nutrient limitation. The fact that the growth inhibition tests are carried out without the
additional stress of nutrient limitation should therefore not disqualify them as a basis for environmental risk
assessment. In any case, studies of growth inhibition under controlled nutrient limitation require the use of
continuous cultures which is a technique demanding too many resources for the purpose of International
Standard toxicity tests.
6.2 pH control
The pH of the test medium affects the toxicity of many chemicals either by its effect on speciation of the
chemicals or by competitive effects of
...
TECHNICAL ISO/TR
REPORT 11044
First edition
2008-12-01
Water quality — Scientific and technical
aspects of batch algae growth inhibition
tests
Qualité de l'eau — Aspects scientifiques et techniques des essais
d'inhibition de croissance d'un lot d'algues
Reference number
©
ISO 2008
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ii © ISO 2008 – All rights reserved
Contents Page
Foreword. iv
Introduction . v
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 General principles of ISO algal growth inhibition tests.2
5 Test species .4
5.1 General.4
5.2 Pseudokirchneriella subcapitata.6
5.3 Desmodesmus subspicatus .9
5.4 Skeletonema costatum.9
5.5 Phaeodactylum tricornutum.12
6 Test conditions .14
6.1 Growth medium.14
6.2 pH control .16
6.3 Inoculum density .18
6.4 Incubation conditions .21
6.5 Test endpoint .23
Bibliography .25
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
In exceptional circumstances, when a technical committee has collected data of a different kind from that
which is normally published as an International Standard (“state of the art”, for example), it may decide by a
simple majority vote of its participating members to publish a Technical Report. A Technical Report is entirely
informative in nature and does not have to be reviewed until the data it provides are considered to be no
longer valid or useful.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO/TR 11044 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2008 – All rights reserved
Introduction
The growth of microalgae in batch cultures follows a well known pattern, with a lag phase followed by an
exponential growth phase, a phase of declining growth rate, a stationary phase, and ultimately a death phase
(Reference [9]). The characteristics of these phases are dependent on the environmental conditions including
the chemical composition of the growth medium, which provides the basis for using batch cultures of algae as
bioassays to investigate growth stimulating or inhibiting properties of constituents of the growth medium.
The first systematic application of microalgae bioassays for which standard protocols were developed was for
assessment of nutrient status and identification of growth limiting nutrients. Skulberg (Reference [50])
developed a bioassay with the green alga Selenastrum capricornutum Printz, which was used to assess
fertilizing influences of pollution in inland waters. The nutrient bioassay with S. capricornutum was further
developed and standardised in Reference [55]. The strain of S. capricornutum used as test organism in the
nutrient bioassays was originally isolated from the river Nitelva in southeast Norway in 1959. It has become
the most commonly used test algae for bioassays and is available from most major culture collections. Due to
taxonomic revisions, it was first renamed Raphidocelis subcapitata and later Pseudokirchneriella subcapitata
(Korshikov) Hindak (Reference [20]).
It was early recognized that bioassays of microalgae could be used to study the growth-inhibiting effects of
toxic chemicals and waste waters, and a modification of the algal assay procedure for toxicity studies was
made in Reference [43]. However, based on compilations of early algae toxicity test data some authors
claimed that the sensitivity of algae generally was low (Reference [26]). The environmental relevance of
results of the tests was also questioned because of the significant interspecies variation in response and lack
of field-validation of results of algal toxicity tests (Reference [28]). On the other hand, microalgae are generally
the most important primary producers in aquatic ecosystems. Excluding the assessment of toxicity to this
group of organisms in risk assessment and environmental management cannot be justified. Development and
standardisation of methods have therefore been undertaken to increase the reproducibility and relevance of
toxicity tests with microalgae. Standardised growth inhibition tests with algae are now a cornerstone in the
environmental management and risk assessment of chemicals. Recent reviews (e.g. Reference [57]) show
that they are often the most sensitive of the “base-set” tests which include also acute toxicity tests with fish
and Daphnia.
In addition to several national organisations, the Organisation for Economic Co-operation and Development
(OECD) and the International Organization for Standardization (ISO) took on the work of developing
guidelines and standards for growth inhibition with microalgae in the late 1970s. The OECD guidelines aim to
test chemical substances, while ISO documents cover tests for composite water samples, such as waste
water and elutriates. However, harmonisation of the procedures was an objective as the two series of
documents were developed in parallel by the two organisations. The development of the freshwater test was
initiated by ISO in 1978. Three ring tests were organised between 1980 and 1982 and included in
ISO 8692:1989, revised as ISO 8692:2004. The first draft of a marine algae inhibition test was produced in
1982, but the first ISO/DIS was not published until 1991, when the method had been ring tested.
ISO 10253:1995 was revised as ISO 10253:2006. In addition to these two standards, ISO 14442:1999,
guidelines for algal growth inhibition tests with poorly soluble matter, volatile compounds, metals and waste
water, was revised as ISO 14442:2006. In this Technical Report, the general principles of the batch culture
growth inhibition tests, and how some critical methological aspects have been addressed in the International
Standards for algal growth inhibition tests, are presented.
TECHNICAL REPORT ISO/TR 11044:2008(E)
Water quality — Scientific and technical aspects of batch algae
growth inhibition tests
1 Scope
This Technical Report discusses scientific and technical aspects that have been considered in connection with
the development of batch algal growth inhibition test procedures specified in ISO 8692, for freshwater, and
ISO 10253, for marine waters.
Previously unpublished results of experiments performed at the Norwegian Institute for Water Research
(NIVA) have been included to demonstrate various aspects.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 8692:2004, Water quality — Freshwater algal growth inhibition test with unicellular green algae
ISO 10253:2006, Water quality — Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum
ISO 14442, Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile
compounds, metals and waste water
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
effective concentration
EC
x
concentration of test sample which results in a reduction of x % in the specific growth rate relative to the
controls
[ISO 8692]
NOTE Unless otherwise stated, the form EC is used in this Technical Report to mean E C where “r” denotes “rate”.
x r x
Effective concentrations based on area under the growth curve can be derived, and these are designated E C , where “b”
b x
denotes “biomass” (see 6.5 for further details).
3.2
specific growth rate
µ
proportional rate of increase in cell density per unit of time:
1dn
µ =
ntd
where
n is the cell density, expressed in cells per millilitre;
t is the time, expressed in days.
NOTE 1 Specific growth rate is expressed in reciprocal days.
NOTE 2 Adapted from ISO 8692.
4 General principles of ISO algal growth inhibition tests
The algae growth inhibition test methods specified in ISO 8692 and ISO 10253 are based on batch cultures
which are inoculated with algae from an exponentially growing inoculum culture and incubated under
continuous illumination. The growth medium, inoculum biomass density, temperature, and illuminance, have
been selected to allow an exponential increase in the algal biomass density during the 72 h incubation period
for the recommended test species.
The experimental design of the tests includes a series of five or more concentrations of the test material in
growth medium prepared in triplicate, and six control replicates without test material. After inoculation with test
algae, the solutions are incubated in transparent, inert containers under continuous illumination and constant
temperature. The cultures should be agitated in order to obtain a homogenous suspension of the algae and to
stimulate gas exchange with the atmosphere. The biomass density in the cultures is measured by direct or
indirect methods at 24 h intervals until termination of the test after 72 h.
An example of a growth inhibition test with Pseudokirchneriella subcapitata is shown in Figure 1. The
substance tested was potassium dichromate. The growth curves show close adherence to exponential growth
in the cultures, and decreasing growth rates with increasing concentration of the test substance. Average
specific growth rates may be calculated as the logarithmic increase in cell density from start to 72 h. Figure 2
shows the concentration/response plot for the endpoint growth rate. A curve has been fitted to the
1)
observations by non-linear regression using a log-logistic model (REGTOX) . Concentrations causing 10 %
and 50 % reduction of the growth rate (EC and EC respectively) have been calculated from the regression
10 50
equation.
1) Available (2008-11-14) at http://eric.vindimian.9online.fr/
2 © ISO 2008 – All rights reserved
Key
1 ρ(K Cr O ) = 0 (control) 5 ρ(K Cr O ) = 1 mg/l
2 2 7 2 2 7
2 ρ(K Cr O ) = 0,25 mg/l 6 ρ(K Cr O ) = 1,6 mg/l
2 2 7 2 2 7
3 ρ(K Cr O ) = 0,4 mg/l n cell density, 10 cells/ml
2 2 7
4 ρ(K Cr O ) = 0,63 mg/l t time, h
2 2 7
Figure 1 — Growth curves (mean values of replicates) for cultures of P. subcapitata
at different mass concentrations of K Cr O
2 2 7
Key
µ specific growth rate as a percentage of control
EC effective concentration at 10 % inhibition
EC effective concentration at 50 % inhibition
ρ(K Cr O ) potassium dichromate mass concentration, mg/l
2 2 7
Figure 2 — Mass concentration/response plot showing the effect of K Cr O
2 2 7
on the growth rate of P. subcapitata
5 Test species
5.1 General
Microalgae constitute a phylogenetically diverse group of organisms, including the procaryotic cyanobacteria
and several phyla of eucaryotic algae. It is therefore not surprising that the sensitivity among different species
of microalgae to various toxic substances is highly variable. Some studies have shown that such interspecies
variation in sensitivity may amount to three to four orders of magnitude (References [2], [24], [54]). This
variation in sensitivity must, of course, be acknowledged when intrepreting data on algal toxicity in a risk
assessment context and the use of a battery of species has been proposed to account for the variation
(References [8], [21], [33], [53]).
ISO 8692 specifies two green algae — P. subcapitata and Desmodesmus subspicatus (previously known as
Scenedesmus subspicatus) — as test species in freshwater. ISO 10253 specifies two marine diatoms,
Skeletonema costatum and Phaeodactylum tricornutum for the marine algae growth inhibition test. A search
for data entries on toxicity of chemicals to the algal species included in the ISO and OECD test methods in the
US EPA database ECOTOX showed a total of approximately 5 000 data entries of which 42 % are from tests
with P. subcapitata, which confirms the position of this strain as a reference alga in bioassays (see Figure 3).
Among the marine species, S. costatum appears to be the one most frequently used.
4 © ISO 2008 – All rights reserved
Key
1 P. subcapitata 5 Navicula pelliculosa
2 Chlorella vulgaris 6 S. costatum
3 D. subspicatus 7 P. tricornutum
4 Anabaena flos-aquae n number of entries
e
a
Freshwater algae.
b
Marine algae.
Figure 3 — Number of data entries on toxicity to algae in the US EPA database ECOTOX
Some characteristics of the ISO 8692 and ISO 10253 test algae are presented in Table 1. The data were
obtained from batch cultures in ISO 8692 (freshwater) and ISO 10253 (sea water) media. The cultures were
2 2)
incubated at 21 °C and continuous illuminance of 80 µmol/m s and analysed in the late exponential phase.
3)
The cell density and mean cell volume were measured using a Coulter Multisizer M3 equipped with a
100 µm orifice tube. The dry mass was measured after collection of the algae on a glass fibre filter which was
dried at 104 °C until constant mass. For the marine species the mass of salts in the water adsorbed in the
filters was corrected for. It should be noted that “cell” in this context refers to particles identified by the particle
counter. For species forming aggregates as e.g. D. subspicatus and S. costatum, the true cell volume and
mass may be less than indicated in Table 1.
2) Both ISO 8692 and ISO 10253 use the term “light intensity” rather than “illuminance”. The photosynthetically available
radiance (PAR) is defined as the total irradiance in the wavelength range 400 nm to 700 nm. Both ISO 8692 and
ISO 10253 indicate in a note that for light-measuring instruments calibrated in the photometric unit, lux, an equivalent
range of 6 000 Ix to 10 000 Ix is acceptable for testing.
3) Example of a suitable product available commercially. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of this product.
Table 1 — Example of size and mass of cells of different ISO test algae
grown in freshwater and marine growth media
Mean cell volume Mean cell dry mass
Species Strain
µm mg
−8
P. subcapitata NIVA/CHL 1 ≡ CCAP 278/4 72 3,0 × 10
−8
D. subspicatus NIVA/CH 55 ≡ SAG.86.81 139
5,3 × 10
−8
S. costatum NIVA/BAC 1 115
4,6 × 10
−8
P. tricornutum
NIVA/BAC 2 56 1,9 × 10
5.2 Pseudokirchneriella subcapitata
P. subcapitata is the most used test alga in growth inhibition tests and is recommended as test species in
several national standards in addition to the international ISO and OECD test protocols. All cultures of this
species maintained in the major culture collections (e.g. CCAP 278/4, ATCC 22662, 61.81 SAG, UTEX 1648)
stem from a clone culture isolated from a Norwegian river in 1959 (Reference [50]). This is a great advantage
from the point of view of reproducibility of test results which is an important aspect of standardisation. The
appearance of P. subcapitata in culture is shown in Figure 4.
a) P. subcapitata
b) D. subspicatus
c) S. costatum
d) P. tricornutum
Figure 4 — Light microscope photographs of cultures of test algae specified
in ISO 8692 and ISO 10253
6 © ISO 2008 – All rights reserved
The cells are solitary and easily counted with an electronic particle counter. Up to eight autospores form within
the cells and are released when the daughter cells are mature. As a result, a non-synchronous culture
contains a mixture of cells of various sizes from small, recently released cells to large cells with visible
autospores. An example of the size distribution of such a culture analysed with a Coulter Multisizer is shown in
Figure 5.
Sometimes, partial synchronisation occurs in toxicity tests with P. subcapitata. In such a case, a size
distribution with two peaks may be observed.
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n V
differential cell number differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 5 — Size distributions of a culture of P. subcapitata
in the exponential growth phase in ISO 8692 growth medium
The requirement for growth of P. subcapitata in the control cultures in ISO 8692 is that the specific growth rate
−1
shall be at least 1,4 d . This corresponds to an increase in cell density of a factor of 67 in 72 h. Normally the
growth rate is well above this requirement under the conditions specified in ISO 8692. Results of an
experiment where cultures of P. subcapitata were incubated on a light/temperature gradient are shown in
Figure 6.
Key Key
2 2
1 14,5 °C 5 29,1 °C 1 30 µmol/m s 5 90 µmol/m s
2 2 2
2 19,6 °C E illuminance, µmol/m s 2 50 µmol/m s 6 100 µmol/m s
−1 2
3 22,7 °C µ specific growth rate, d 3 70 µmol/m s T temperature, °C
2 −1
4 25,6 °C 4 80 µmol/m s µ specific growth rate, d
NOTE The range of illuminance specified in NOTE The range of temperature specified in ISO 8692
ISO 8692 is indicated by the frame inside the figure. is indicated by the frame inside the figure.
a) plotted against illuminance b) plotted against temperature
Figure 6 — Effects on the growth rate of P. subcapitata in ISO 8692 growth medium
In this experiment, the cell density was measured twice every day for 5 d, and the growth rate calculated by
regression analysis of cell density against time for the exponential part of the growth curve. The results
indicate that the maximum growth rate occurs at a temperature of approximately 32 °C. In the temperature
range specified in ISO 8692 (21 °C to 25 °C) a linear increase of growth rate with temperature was observed.
2 −1
At an illuminance of 90 µmol/m s, the growth rate increased by 0,16 d for each degree celsius between
2 −1 −1
17 °C and 27 °C (R = 0,995 1). This means that the growth rate increased from 1,38 d at 21 °C to 2,02 d
at 25 °C.
The effect of illuminance was highly temperature dependent. At temperatures below 20 °C, the growth rate
2 2
was almost unaffected by illuminance in the range 30 µmol/m s to 100 µmol/m s. At higher temperatures,
−1
the growth rate increased with illuminance, but even at 22,7 °C, the growth rate increased only 0,2 d
2 2
between 60 µmol/m s and 100 µmol/m s. The growth response at higher temperatures indicates that growth
is saturated at about 90 µmol/m s. The results are in agreement with Reference [35], which reports growth
−1 2 −1 2
rates of approximately 1,31 d at 50 µmol/m s and 1,51 d at 100 µmol/m s at a temperature of 20,8 °C.
8 © ISO 2008 – All rights reserved
5.3 Desmodesmus subspicatus
Different strains of D. subspicatus can be obtained from the major culture collections. The strain specified in
ISO 8692 (SAG 86.81 ≡ CCAP 276/22 ≡ UTEX 2594) was originally isolated by Brinkmann from an aquarium
in Berlin in 1953. According to Reference [17], this strain grows mainly as single cells in culture. It may,
however, also occur in colonies (coenobia) of four cells to eight cells arranged in a row or as loose aggregates
without systematic organisation [see Figure 4 b)]. Cell length and width reported in Reference [17] are 3,5 µm
to 5,0 µm and 4,0 µm to 6,0 µm, respectively. The size distribution of a culture in ISO 8692 medium is shown
in Figure 7.
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n differential cell number V differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 7 — Size distributions of a culture of D. subspicatus in the exponential growth phase
in ISO 8692 growth medium
The size distribution plots indicate that the single cells with a spherical diameter of approximately 4 µm to
7 µm dominate. The long tail in the distributions with spherical diameters up to at least 15 µm, which is
especially pronounced in the differential volume plot, is probably caused by the presence of aggregates.
5.4 Skeletonema costatum
Several strains under the name of S. costatum are available in the major culture collections. In ISO 10253,
two strains are specified: CCAP 1077/1C (origin: North Sea, post-1970) and NIVA BAC 1 (origin: Oslo Fjord,
1962). In the CCAP strain catalog, two other strains (CCAP 1077/3 and 1077/5) are listed as strains used for
ecotoxicity testing, while this is not the case for CCAP 1077/1C. Probably several different strains are used as
test organisms. The issue is further complicated by recent taxonomic revisions which have revealed that
S. costatum is a “species complex” rather than a true species, and several previous “S. costatum” strains have
been renamed as S. grethae (e.g. CCAP 1077/3 and 1077/4) or S. pseudocostatum (CCAP 1077/7) (see
Reference [49]). Apparently CCAP 1077/1C has not yet been examined for taxonomic revision. The stain
NIVA BAC 1 has recently been examined and allocated to S. pseudocostatum.
The size distribution of the strain NIVA BAC 1 grown in ISO 10253 medium is shown in Figure 8. The
distribution shows a fairly narrow peak around 6 µm, indicating that the culture is predominantly single celled
or that the particle counter is able to distinguish each cell even if they are connected in chains [see
Figure 4 c)].
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n differential cell number V differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 8 — Size distributions of a culture of S. costatum strain NIVA BAC 1 (S. pseudocostatum)
in the exponential growth phase in ISO 10253 growth medium based on natural sea water
During the development of ISO 10253, a comparison of different S. costatum strains was performed
(Reference [13]). The strains included were ISTPM P4, NIVA BAC 1, CCAP 1077/1C, CCAP 1077/3, and
CCAP 1077/5. The study showed differences in cell size and chain length between the strains and suggested
that these differences were correlated to differences in sensitivity to the reference substances potassium
dichromate and 3,5-dichlorophenol (DCP). The results are summarized in Table 2.
Table 2 — Results of tests of reference substances with different strains of S. costatum
Potassium dichromate 3,5-Dichlorophenol
Control growth Mean particle size in
rate control
EC EC
Strain
50 50
−1
mg/l mg/l µm /cell
d
ISTPM P4 6,6 1,4 2,64 to 3,36 563
NIVA BAC 1 2,6 0,95 1,68 to 2,11 120
CCAP 1077/1C 3,9 1,2 1,65 to 1,94 169
CCAP 1077/3 7,4 1,4 2,64 to 2,40 596
CCAP 1077/5 5,7 1,3 2,25 to 2,40 454
In future revisions of ISO 10253, the implications of the recent taxonomic revision of the “Skeletonema
costatum complex” should be considered. Preferably only one strain of S. costatum should be recommended
as test alga.
10 © ISO 2008 – All rights reserved
The effect of temperature on the growth rate of strain BAC 1 in ISO 10253 growth medium based on natural
sea water is shown in Figure 9. The specific growth rate was calculated from the increase in biomass density
(measured as total cell volume with a Coulter Multisizer) in batch cultures after 70 h incubation at
80 µmol/m s. The results showed an almost linear increase in growth rate with temperature in the range
−1 2
13 °C to 21 °C. Linear regression of the data show a slope of 0,141 d per degree celsius (R = 0,996 2). The
optimum temperature for this strain appears to be around 24 °C and growth was completely inhibited at
32,5 °C. It should be noted that the growth rates observed in this experiment are somewhat lower than what is
normally found in control cultures in toxicity tests with the same strain under similar conditions. The specific
growth rates obtained in control cultures incubated at approximately 75 µmol/m s and 21 °C are usually
−1
around 2,0 d . The low calculated growth rates in the experiment are probably due to the fact that the growth
rate was not exponential throughout the incubation period.
Key
T temperature, °C
−1
µ specific growth rate, d
Figure 9 — Effect of temperature of average growth rate (0 h to 70 h) of S. costatum (NIVA BAC 1)
incubated in ISO 10253 natural sea water medium at 80 µmol/m s
Because of the high growth rate of most S. costatum strains (see Table 2), exponential growth is often not
maintained for 72 h under the specified test conditions, even if the inoculum density is held at 5 × 10 cells/ml.
This may have the effect that partly inhibited cultures catch up with the control cultures during the last day of
the test as shown in Figure 10 a). In this example, the significant growth inhibition that was observed in 1 mg/l
of 3,5-DCP after 48 h was no longer seen after 72 h. Deviation from exponential growth of S. costatum was
noted also in the ISO and PARCOM ringtests (References [1], [15]). It is obvious that it is more appropriate in
this case to base the calculations of growth rates on the first 48 h of the test when the growth in the control
cultures is still exponential. In ISO 10253:2006, such a recommendation was included.
Key Key
1 ρ(3,5-DCP) = 0 (control) 5 ρ(3,5-DCP) = 2,5 mg/l 1 ρ(3,5-DCP) = 0 (control) 5 ρ(3,5-DCP) = 4 mg/l
2 ρ(3,5-DCP) = 0,63 mg/l 6 ρ(3,5-DCP) = 4 mg/l 2 ρ(3,5-DCP) = 1 mg/l
3 3
3 ρ(3,5-DCP) = 1 mg/l n cell density, 10 cells/ml 3 ρ(3,5-DCP) = 1,6 mg/l n cell density, 10 cells/ml
4 ρ(3,5-DCP) = 1,6 mg/l t time, h 4 ρ(3,5-DCP) = 2,5 mg/l t time, h
a) S. costatum (NIVA BAC 1) b) P. tricornutum (NIVA BAC 2)
Figure 10 — Growth curves obtained from 3,5-dichlorophenol toxicity tests
5.5 Phaeodactylum tricornutum
The strains of P. tricornutum specified in ISO 10253 are CCAP 1052/1A (origin: off Plymouth, UK) and
NIVA BAC 2. The origin of BAC 2 is not known. It was obtained from the University of Oslo and it may be
identical to CCAP 1052/1A. Size distributions of a culture of the strain BAC 2, grown in ISO 10253 medium
based on natural sea water, are shown in Figure 11. P. tricornutum appears as single cells in culture and the
variation in cell size is usually small. Typical growth rates of this strain in ISO natural sea water medium are
−1 2
around 1,6 d at 21 °C and 75 µmol/m s.
From a technical point of view, P. tricornutum is an excellent test organism (low variation in cell size, does not
form aggregates, intermediate growth rate). In contrast to S. costatum, exponential growth is usually
maintained for 72 h under the test conditions specified in ISO 10253 [see Figure 10 b)]. In spite of this,
P. tricornutum has been used less frequently than S. costatum in marine algae toxicity tests (Figure 3). The
main reason for this is probably that S. costatum is considered as a more relevant species from an ecological
point of view. Furthermore, the requirement to use S. costatum as a test organism in the PARCOM test
programme for offshore chemicals has contributed to its popularity. Nevertheless, P. tricornutum was included
in a ring test organised by PARCOM in 1991. In comparison to S. costatum, P. tricornutum was less sensitive
to the reference substance 3,5-DCP (which is in agreement with the result of the ISO ring test) but more
sensitive to two offshore chemicals (Reference [1]).
P. tricornutum inhabits brackish waters and has a wider salinity tolerance than S. costatum. It is therefore a
suitable species for tests in brackish waters or for testing toxic effects of “fresh” waste waters in sea water. By
selection of different species specified in ISO 8692 and ISO 10253, toxicity tests may be performed at all
salinities from freshwater to sea water. Figure 12 shows the effect of salinity on the growth rate of
P. subcapitata, P. tricornutum (NIVA BAC 2), and S. costatum (NIVA BAC 1) in ISO media prepared with
different proportions of distilled water and natural sea water.
12 © ISO 2008 – All rights reserved
Key Key
d size as spherical diameter, µm d size as spherical diameter, µm
n differential cell number V differential cell volume
c c
a) by differential cell number b) by differential cell volume
Figure 11 — Size distributions of a culture of P. tricornutum strain NIVA BAC 2 in the exponential
growth phase in ISO 10253 growth medium based on natural sea water
Key
−1
1 P. subcapitata µ specific growth rate, d
2 P. tricornutum ρ(NaCl) salinity (sodium chloride mass concentration), g/l
3 S. costatum
Figure 12 — Growth of P. subcapitata, P. tricornutum (NIVA BAC 2),
and S. costatum (NIVA BAC 1) at different salinities
6 Test conditions
6.1 Growth medium
The composition of the growth medium may affect the toxic response of the test algae, either because of
chemical interactions that affect the biological availability of the test substance or because of its effects on the
physiological status of the algae. Influence of the growth medium composition on the expression of toxicity in
algal tests has in particular been demonstrated in tests of toxic metals, where pH, hardness and presence of
chelating agents are important factors (Reference [22]). Of course, the same factors affect the toxicity to algae
in their natural environments. A standardised growth medium can therefore not be representative to all natural
environments, a fact that must be recognised when interpreting toxicity data from tests with algae as well as
for other aquatic organisms.
Test media for the ISO growth inhibition tests have been designed to allow unrestricted exponential growth of
the recommended test algae under the specified culturing conditions, avoiding as much as possible excessive
concentrations of ingredients that may cause chemical interference with various test substances. For the
freshwater test (ISO 8692) a specific growth medium was developed after experience from early ring tests
indicated that differences in composition of the medium affected the test results. It was also concluded that a
high buffering capacity was required in order to reduce pH variations which were identified as one of the main
reasons for deviations in results between different laboratories (Reference [13]). In the third ISO ring test, a
−
medium buffered with 150 mg/l NaHCO and NO nitrogen source were used. Still, a substantial pH increase
was observed after 96 h incubation (Reference [14]). Later it was decided to restrict the exposure to 72 h, and
modifications of the medium were made, including lowering the concentration of NaHCO to 50 mg/l and
− +
replacing K HPO with KH PO . NO was replaced with NH as a nitrogen source in order to counteract pH
2 4 2 4 3 4
increase that tended to occur towards the end of the test when the CO demand from algal photosynthesis
exceeded the mass transport of CO across the water/gas interface.
Chelating agents are required to avoid precipitation of iron in the growth medium. However, chelators may
reduce the availability and effect of toxic metals. EDTA (ethylenediaminetetraacetate) is the most commonly
used chelator in artificial algal growth media. In ISO 8692:1989, the medium contained 80 µg/l FeCl ·2H O
3 2
and 100 µg/l of Na EDTA·2H O. Later, the iron content was reduced to 64 µg/l of FeCl ·2H O in order to
2 2 3 2
obtain equimolar concentrations of EDTA and complexing trace metals. Still the medium is not optimal for
testing metal toxicity. Different modifications of the medium have been used to overcome this shortage. In
Reference [18], 32 µg/l humic acid replaced EDTA in order to create a medium with metal complexing
properties more similar to natural surface waters. Some reported tests (e.g. Reference [31]) employed
synthetic growth media without organic chelators. Experience has shown, however, that the growth of algae is
highly variable in such media. Iron is required as a trace element in the medium, and the concentration
required to support the production of biomass in a 72 h test is above the solubility of iron(III) hydroxide. The
effect of excluding EDTA in the medium is demonstrated in Figure 13, which shows the growth performance of
P. subcapitata in the ISO medium with and without EDTA. The two media were inoculated with 5 × 10
cells/ml of P. subcapitata grown in medium with EDTA. The average specific growth rate in six cultures with
−1
EDTA was 1,68 d with a coefficient of variation of 2,9 %. In nine cultures without EDTA, the average growth
−1
rate was 1,15 d with a coefficient of variation of 21 %.
Studies performed at the Danish Technical University demonstrated that the concentration of iron and EDTA
could be reduced without affecting the growth performance of P. subcapitata and based on this finding, a
recommendation for a modified medium for testing of metals was included in ISO 14442, a guidance
document for tests with poorly soluble materials, volatile compounds, metals and waste water. The proposal is
to use 20 µg/l (0,074 µmol/l) of FeCl ·2H O and 22,6 µg (0,083 µmol/l) Na EDTA·2H O in the medium and to
3 2 2 2
reduce the test duration from 3 d to 2 d.
14 © ISO 2008 – All rights reserved
Key
1 with EDTA
2 without EDTA
n
cell density, 10 cells/ml
t
time, h
Figure 13 — Growth of P. subcapitata in ISO 8692 growth medium with EDTA (0,27 µmol/l)
and without EDTA (mean values and standard deviation)
The ISO 10253 marine test can be performed in synthetic or natural seawater of (30 ± 5) g/kg salinity. The sea
water is spiked with nutrients in order to support exponential growth of the test algae for 72 h. The nutrient
[58]
medium has been adopted from ASTM E 1218 . Because of the high content of anions in the sea water, a
higher concentration of EDTA than in the freshwater medium is required to avoid precipitation of iron. During
the development of the ISO 10253 test, some work was done to investigate the possibility of reducing the
content of EDTA in the medium in order to make it more suitable for testing metals. Experiments at NIVA
performed in 1991 showed that the BAC 1 strain grew as well in a natural sea water medium with iron and
EDTA reduced to the levels used in the freshwater medium (ISO 8692) provided that the zinc concentration
was also reduced from 150 µg/l to 15 µg/l. Reduced concentrations of iron and EDTA were also used by
several laboratories that participated in a ring test organised by PARCOM in 1991 (Reference [1]). Some
laboratories reported, however, that S. costatum would not grow as well in the modified medium as in the
original ISO 10253 medium. This may be due to the differences in the composition of the natural sea water
used by different laboratories and the fact that different strains of S. costatum may have different nutrient
requirements. It was found that any modifications of the ISO 10253 medium would need to be based on
extensive trials in many laboratories and the original formulation with 149 µg/l iron and 15 mg Na EDTA·2H O
2 2
has been retained.
Since the growth inhibition tests have been designed to allow exponential growth in a batch culture, the algae
will not experience nutrient limitation. This is different from the natural habitats of most microalgae, where the
growth is often limited by the supply of a limiting nutrient. It has been argued that the stress from nutrient
limitation may increase the sensitivity of algae to toxic substances and some studies indicate that this may be
the case for the effects of metals on P. subcapitata (References [3], [30]). Other studies have also shown that
effects on nutrient uptake by algae may occur at lower concentrations of toxicants than those affecting growth
(References [38], [46]), and such effects may have important consequences for the competitive ability of algae
in a nutrient-limited environment. However, it may also be argued that the potential for a toxicant to cause a
significant effect on the growth of an alga is higher when the growth rate is high than when it is reduced as a
consequence of nutrient limitation. The fact that the growth inhibition tests are carried out without the
additional stress of nutrient limitation should therefore not disqualify them as a basis for environmental risk
assessment. In any case, studies of growth inhibition under controlled nutrient limitation require the use of
continuous cultures which is a technique demanding too many resources for the purpose of International
Standard toxicity tests.
6.2 pH control
The pH of the test medium affects the toxicity of many chemicals either by its effect on speciation of the
chemicals or by competitive effects of hydrogen ions on the binding to the uptake sites. Ionisable organic
substances such as chlorophenols are more lipophilic and exert a higher toxicity in their un-ionised than in
their ionised form (Reference [51]). For metals, the effect of pH may be very complicated since it may affect
both the forming of complexes of different biological availability and the binding at the cell surface ligands
(References [5], [18], [45]).
In batch algal cultures as well as in the natural environments of plankton algae, the photosynthetic activity of
the algae affects the pH of the aquatic environment through displacement of the carbonate system. The algae
assimilate CO , dissolved in the water as a carbon source for photosynthesis. The consumption of dissolved
CO will trigger diffusion of CO from the atmosphere to restore the equilibrium concentration. In a batch
2 2
culture, the CO demand increases with the biomass. When the rate of CO consumption exceeds the mass
2 2
−
−
transfer of CO through the water surface, CO will be derived from HCO which results in liberation of OH
2 2 3
and an increase in pH. The mass balance of carbon in algal batch cultures has been described in
Reference [12].
−
The ISO 8692 freshwater medium is mainly buffered by HCO . The content of NaHCO (50 mg/l) gives a pH
3 3
of approximately 8,1 when the medium is in equilibrium with CO in the atmosphere. When inoculated with
algae and incubated as described in ISO 8692, the p
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