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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer reviewed paper using methodology similar to recognised methods (Algae done in line with EPA method). No purity or GLP data.
Qualifier:
according to
Guideline:
other: EPA 600/9-78-018
Deviations:
not specified
Principles of method if other than guideline:
Method reference:
EPA, The, Selenastrum capricornutum Printz algal assay bottle test. Experimental design, application, and data interpretation. Protocol EPA 600/9-78-018. Corvallis Environ. Research Laboratory; Office of Research and Development, Corvallis, Oregon: 126 pp.
GLP compliance:
not specified
Analytical monitoring:
not specified
Details on sampling:
- Concentrations: 64, 32, 16, 8, 4 and 2 µg.l-1
- Sampling method: For the toxicity tests the alga inoculum (1 ml) was taken from a preculture in exponential growth phase (6th day of growth).
Vehicle:
not specified
Details on test solutions:
The standard solution of the test material was prepared by dissolving the test material in bidistilled water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Selenastrum capricornutum
- Source (laboratory, culture collection): CCAP (The culture collection of Algae and Protozoa; Institute of Freshwater Laboratory, Far Sawrey, Ambleside, Cumbria LA22 OLP,.UK).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
5 d
Post exposure observation period:
No data
Hardness:
No data
Test temperature:
20 ± 1 °C
pH:
No data
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
64, 32, 16, 8, 4 and 2 µg/L
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 mL solutions
- Initial algal density: 5 x 10ˆ5 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: AFNOR medium

The alga cultures were incubated at 20 ± 1 °C in AFNOR medium with an photoperiod of 8 h dark and 16 h light and with an illumination of about 4000 lumens. For the toxicity tests the alga inoculum (1 mL) was taken from a pre-culture in exponential growth phase (6th day of growth).
The initial algal density was 5 x 10^5 cells/mL. The culture medium was used as dilution water and for the control.
The tested concentrations were the following: 64, 32, 16, 8, 4 and 2 µg/L. The test algae were cultivated in 100 mL solutions containing the test material with two replicates in the preliminary test and three replicates in the definitive tests, for each concentration and for the control.
The growth was verified daily by counting the number of cells per unit volume through a Zeiss microscope in a counting chamber (Buerker Chamber).
Cell densities were reported as the mean of 4 counts; the coefficient of variation of this technique was 2.5 %. The results were expressed as percent inhibition of the algal growth and the EC50 value, 95 % confidence interval and NOEC value were calculated by Probits Analysis. The replicate data were analysed by the analysis of variance (ANOVA), and the toxicity effects at different concentrations were compared by Bonferroni test.

Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
12.4 µg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL: 11.0-13.9 µg/L
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.2 µg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
In order to verify the effects of the test material on Selenastrum capricornutum three tests were undertaken: one preliminary and two definitive ones.
The preliminary test already revealed a marked inhibiting effect on the growth at 8 µg/L; an effect which was more and more noticeable reaching 100 % inhibition at a concentration of 64 µg/L.
In the first definitive test the concentrations analysed were 2, 4, 8 and 64 µg/L. At 2 no effect was observed, whereas at 4 µg/L a partial stimulus effect both in duplication and in cellular growth was observed.
At 8 and 64 µg/L, on the other hand, an inhibiting effect on growth was evident.
In the second definitive assay the tested concentrations were 4, 8, 16 and 32 µg/L. In Figure 3 the algal growth rate at different test material concentrations is reported.
The obtained data confirmed the stimulus effect at 4 µg/L: this effect was also confirmed statistically with the Bonferroni test. Furthermore the range of concentrations utilised permitted the calculation of EC50 value, that is the toxicant concentration halving the algal growth rate, with the relative 95 % confidence interval and NOEC value.
The value achieved of EC50 at 96 h was of 12.4 µg/L with 95 % confidence interval of 11.0 - 13.9 µg/L. The NOEC value was 1.2 µg/L.
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
No data
Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of this study the EC50 of the test material at 96 h was 12.4 µg/L with 95 % confidence interval of 11.0 - 13.9 µg/L. The NOEC value was 1.2 µg/L.
Executive summary:

The obtained data confirmed the stimulus effect at 4 µg/L: this effect was also confirmed statistically with the Bonferroni test. Furthermore the range of concentrations utilised permitted the calculation of EC50 value, that is the toxicant concentration halving the algal growth rate, with the relative 95 % confidence interval and NOEC value.

The EC50 at 96 h was of 12.4 µg/L with 95 % confidence interval of 11.0 - 13.9 µg/L. The NOEC was 1.2 µg./L.

Description of key information

In the key study for acute toxicity to algae (Miana, 1993) the study was conducted according to the EPA 600/9-78-018 method using the species, Selenastrum capricornutum. The value achieved of EC50 at 96 h was of 12.4 µg/L with 95 % confidence interval of 11.0 - 13.9 µg/L. The NOEC value was 1.2 µg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
12.4 µg/L
EC50 for marine water algae:
0.26 µg/L
EC10 or NOEC for freshwater algae:
1.2 µg/L

Additional information

In the key study (Miana et al, 1993), the study was conducted according to the EPA 600/9-78-018 method that is comparable to OECD 202 and a standard species, Selenastrum capricornutum, were used.

The reliability rating for this study is 2, according to the criteria of Klimisch, 1997 as this is a peer reviewed paper using methodology similar to recognised methods, however no purity or GLP data was presented. This study is considered the most relevant and reliable study for this endpoint, however all of the following supporting studies correlate with this key study and the values are all <1 mg/L which is the cut-off value used for classification purposes for this endpoint.

In the Arrhenius et al (2006) paper, effects on periphyton community photosynthesis and reproduction of the unicellular green algae Scenedesmus vacuolatus were investigated in a 24 h hour acute toxicity experiment.

The test material was highly toxic in the algal test, with EC50 and NOEC values of 215 and 133 nM respectively. Taking into account the molecular weight of the substance (325.51), the EC50 and NOEC concentrations are 70.0 and 43.3 µg/L.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented. Also this is a non-standard study using a non-standard species.

In the Arrhenius et al (2006) paper, periphyton studies were conducted at Kristineberg Marine Research Station by the Gullmar fjord on the Swedish west coast. Stock solutions of the test material were prepared in methanol for the algal reproduction test and stored at -20 °C. The test material was highly toxic in the periphyton test, with EC50 and NOEC values of 59 and 32 nM respectively.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented. Also this is a non-standard study using a non-standard species.

In the Blanck et al (1984) paper, 13 algal strains were examined in growth inhibition experiments using the test material.

The median EC100 given for the test material based on 13 species of algae is 0.13 mg/L.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methodolgy is well documented. Also this is a non-standard study.

In the Walter et al (2002) paper, liquid cultures of the unicellular green alga Scenedesmus vacuolatus were exposed to the test material in order to examine the inhibition of cell growth caused by the test material. The EC50 given for the test material for Scenedesmus vacuolatus is 0.270 µmol/L. The NOEC was found to be 0.113 µmol/L based on inhibition of algal cell reproduction.

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as there was limited information on test material and no information on GLP, no guideline was specified and the methods and results are non-standard.

In the Wong et al (1982) paper, primary productivity was measured by the amount of 14C-carbonate taken up by algae over a 4 hour period. After a further 24 hours incubation, the level of inhibition was determined.

The primary productivity IC50 values for A. falcatus, S. quadricauda, A. flos-aquae and Lake Ontario algae PP were 0.020, 0.016, 0.013 and 0.003 mg/L respectively. The inhibition of reproduction IC50 value for A. falcatus was 0.005 mg/L.

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as there was limited information on test material and no information on GLP, no guideline was specified and a vehicle was used.

In the Walsh et al (1985) paper, two methods were used for estimation of effects of tin compounds on algae. The first was a test of algal population growth and the second was designed to estimate concentrations of organotins that were lethal to S. costatum.

The EC50 of the test material was 0.26 µg/L when tested with S. costatum for growth rate.

The EC50 and LC50 for tributyltin chloride on cell death of S. costatum are 5.6 and 11.5 µg l-1 respectively.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the the methods are well described and peer-reviewed. This study is considered the most relevant and reliable study conducted under saltwater conditions, therefore the 72 hr result will be used as the key value for these conditions.

In the Huang et al (1996) paper, the inhibition by organotins of the growth of the algae was determined in a 96 hour test in saltwater using Scenedesmus obliquus and Platymonas sp.

The EC50 values for S. Obliquus and Playmonas sp. exposed to the test material are 0.58 and 0.31 ng Sn/L respectively. EC50 values of the test material for the two algae are less than 1 ppb. The test material is highly toxic to algae.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although there was limited information on test material and no information on GLP, the methods are well described.