Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3-6 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
yes
Remarks:
Uncritical deviations: experiment temperature range 20.7 – 22.2 °C, pre-culture incubation range 19.0 – 23.3 °C, autoclaved for 20 instead of 15 min, pre-culture preparation 3-4 days before test start
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
as above
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: The content of the test item was estimated in the test solutions at the start and the end (72 h) of the test

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The water-accommodated fractions (WAF) were prepared for the test. This was done by mixing the real loads of 1 / 3.2 / 10 / 32 / 100 mg/L resp. 1.1 / 3.5 / 10.9 / 34.8 / 108.7 µL/L test item (based on a density of 0.92 g/mL stated in the SDS) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring moderate for 24 hours at 120 rpm on a magnetic stirrer. No turbidity of the test solution could be observed. The lower phase was used unfiltered as test solutions.
- Controls: Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: 86.81
- Source: The algae culture was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared.
For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 19.0 – 23.3°C; see chapter 13). The result-ing culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
not applicable

Test conditions

Hardness:
no data
Test temperature:
20.7 – 22.2°C
pH:
7.6-8.6
Dissolved oxygen:
no data
Salinity:
not applicable
Conductivity:
no data
Nominal and measured concentrations:
1 / 3.2 / 10 / 32 / 100 mg/L loading rate
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Material, size, headspace, fill volume: The test vessels were filled with 45 ± 1 mL
- Initial cells density: 2500 cells/mL
- Control end cells density: 531507 Cell Number/mL
- No. of vessels per concentration (replicates): 3 replicates for each treatment
- No. of vessels per control (replicates): 6 replicates for the blank control

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: within the specified range (4440 – 8880 Lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter every 24h, observation of cell abnormalities

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Results used to determine the conditions for the definitive study: The concentrations to be tested are based on non GLP pre-tests
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) in a separate reference test

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): microscopical observations confirmed the normal and healthy appearance of the algae
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The concentrations measured in the test solutions are in the same very low range as the blank value. It is detectable but not quantifiable. In addition, the test solutions were in direct contact with the test item during stirring. This means that the soluble components must have dissolved. Therefore, the results are related to the loading rate
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72h ErC50 0.66 mg/L (95% C.I. 0.64 – 0.69 mg/L); 72h EyC50 0.40 mg/L (95% C.I. 0.38 – 0.41 mg/L). The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).
Reported statistics and error estimates:
As no inhibition could be observed no statistical evaluation was performed

Any other information on results incl. tables

A slight inhibition of algal growth was observed only in the lowest concentration (1 mg/L). However, this is probably due to contamination or carry-over, as all higher concentrated treatments do not show any inhibition.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure to test item of Desmodesmus subspicatus for 72 h did not induced any inhibition, determining an NOELR and EL50 for growth and yield greater than 100 mg/L (loading rate).
Executive summary:

A study was performed in a Water Accommodated Fraction (WAF) test setup exposing the algae,Desmodesmus subspicatus, for 72 h to concentration range of 1.0 to 100 mg/L (loading rate) of the test item. Test was performed according to the OECD guideline 201 and it fulfilled the validity criteria. Measured concentrations were estimated by by determination of the total organic carbon (TOC) content in the test solutions using a carbon analyser. The concentrations measured in the test solutions are in the same very low range as the blank value. It is detectable but not quantifiable. Therefore, the results are related to the loading rates; see Guidance Doc. No.23 (§153). No inhibition was observed after 72 h, determining a NOELR, LOELR, EL10 and EL50 for growth and yield greater than 100 mg/L (loading rate).