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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:
2013-03-26 to 2013-03-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
24 August 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
23rd March, 2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
April 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentration, samples were taken from the testing concentration and the control at the start and at the end of the test. Four replicates samples were taken at the start and at the end of the study from the test solution, and two samples were taken from the control samples.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Formulation:
The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent. The test solution was freshly prepared at the beginning of the experiment, in the testing laboratory.

- Dosage of test item:
In order to obtain the saturated test solution water-accommodated fractions of the test item (WAF method, according to OECD Series on Testing and Assessment No. 23) were prepared as follows:
A supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving the test item amount into the test medium (OECD medium) one day before the start of the test. This solution was moderately stirred for a period of approx. 24 hours and then left settled for about 1 hour. After settling the non-dissolved test material was separated by 20 min centrifugation at 5000 rpm and 20°C in order to obtain the saturated test solution.

- Untreated control:
The dilution water (without addition of the test item) was used as a test solution.


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Origin: The algae were supplied by the SAG: Collection of Algal Cultures, D-37073 Göttingen, Germany
- Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days at this test.)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.1 – 22.3 °C
pH:
7.93 – 9.23
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
No measured concentrations (below LOQ)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium. Each test unit was uniquely identified with study number, test item concentration and replicate number.
- Initial cells density: The initial cell density was about 104 cells/mL in each test flask.
- No. of vessels per concentration: The test was performed with six replicates per test concentration.
- No. of vessels per control (replicates): The test was performed with six replicates in the untreated control group.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light Intensity:
The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks during the test was 7691 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED
- Determination of cell number: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.

TEST CONCENTRATIONS
- Concentration (nominal): 100 mg/L
- Range finding study and results used to determine the conditions for the definitive study:
A preliminary test was performed at aquatic saturation to check that the test item has no influence on the growth of algae up to its solubility limit in the test medium (saturation concentration). In order to obtain the saturated test solution water-accommodated fractions of the test item (WAF method, according to OECD Series on Testing and Assessment No. 23) were prepared as follows: A supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving the test item amount into the test medium (OECD medium) one day before the start of the test. This solution was moderately stirred for a period of approx. 24 hours and then left settled for about 1 hour. After settling the non-dissolved test material was separated by 20 min centrifugation at 5000 rpm and 20°C in order to obtain the saturated test solution. The pH of the obtained test solution was not adjusted and was determined to be 7.59. Algal cells were exposed to the test concentration plus a control, for 72 hours. The test was performed with three replicates per test concentration and control group. Significant toxic effects were not observed during the preliminary test, therefore only one test concentration at saturation and one control group were tested in a limit test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: No
- Cell density in control: The cell density in the control has increased from nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to N = 64.17 × 104 cells/mL (mean value) after 72 hours of exposure. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
Results with reference substance (positive control):
The date of the last study with the reference item Potassium dichromate was: 11 – 14 March 2013.
The 72h ErC50: 1.40 mg/L, (95 % confidence limits: 1.27 – 1.54 mg/L)
The 72h EyC50: 0.78 mg/L, (95 % confidence limits: 0.70 – 0.87 mg/L)
Reported statistics and error estimates:
Statistical analysis:
Mean values and standard deviations were calculated using Excel for Windows software.
Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using 2 Sample t-Test (alpha = 0.05) by TOXSTAT software. The ErCx and EyCx values of the test item were determined from the raw data.

Analytical results:

Concentration of the test item both in the test and control group were below the quantification limit (LOQ = 5 mg/L) considering tenfold concentration of the samples before the analysis. Therefore, nominal concentrations were used.

Biological results

Growth inhibition: Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software. The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield at the treatmentgroup (100 % v/v saturated solution, equivalent to 100 mg/L nominal concentration) was not statistically significantly different from the untreated control. Accordingly, the No Observed Effect Concentration (NOEC) was determinedas the saturation concentration (equivalent to 100 mg/L nominal concentration). The 72 h-EC50 values based on growth rate and yield were determined to be higher than saturation.

Microscopic examinations:

Shape of the algal cells: Abnormal appearance of the algal cells was not observed during the experiment.

Cell Density in Control: The cell density in the control has increased from nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to N = 64.17 × 104 cells/mL (mean value) after 72 hours of exposure. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.

Validity criteria fulfilled:
yes
Conclusions:
In this 72-h algal growth inhibition test with Pseudokirchneriella subcapitata the EC50 and the NOEC (both based on growth rate and yield) were determined to be > 100 mg/L and 100 mg/L, respectively.
Executive summary:

This 72-h algal growth inhibition test with Pseudokirchneriella subcapitata was performed in accordance with OECD no. 201 guideline, EU method C.3 and OPPTS 850.5400 guideline. The obtained results showed that the test item ditetradecyl peroxydicarbonate had no toxic effect at aquatic saturation (i.e. 100% v/v saturated solution, equivalent to 100 mg/L nominal concentration) on the algae. Accordingly, the EC10, EC50 and the LOEC values based on growth rate and yield were determined to be higher than the solubility level of the test item in the test medium which corresponds to a nominal value of > 100 mg/L. The NOEC was determined to be 100 mg/L (nominal concentration).

Description of key information

In a 72-h algal growth inhibition test with Pseudokirchneriella subcapitata the EC50 and the NOEC (both based on growth rate and yield) were determined to be > 100 mg/L and 100 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

This 72-h algal growth inhibition test with Pseudokirchneriella subcapitata was performed in accordance with OECD no. 201 guideline, EU method C.3 and OPPTS 850.5400 guideline. The obtained results showed that the test item ditetradecyl peroxydicarbonate had no toxic effect at aquatic saturation (i.e. 100% v/v saturated solution, equivalent to 100 mg/L nominal concentration) on the algae. Accordingly, the EC10, EC50 and the LOEC values based on growth rate and yield were determined to be higher than the solubility level of the test item in the test medium which corresponds to a nominal value of > 100 mg/L. The NOEC was determined to be 100 mg/L (nominal concentration).

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