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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 100 mg/L (nominal)
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.

Test solutions

Vehicle:
no
Remarks:
medium was used
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To produce the only test item concentration, 200.9 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 µm. The pH was measured to be 4.7 and was adjusted to pH 8.0.
100 mL of the solution were taken and 0.462 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 6 replicates were prepared. All flasks were sealed with cotton stoppers.
- Controls: medium without any test item
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): see attachment 4
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 μm.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).

ACCLIMATION
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a Cellcounter Z2, Beckman Coulter.
- Culturing media and conditions (same as test or not): Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (attachment 4).
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium (attachment 4) to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
Water hardness of the final nutrient medium was 1.3 °dH, corresponding to 22.5 mg/L CaCO3.
Test temperature:
21 - 24 °C
pH:
The pH was measured to be 4.7 and was adjusted to pH 8.0.
Nominal and measured concentrations:
Nominal: 100 mg/L
Measured: 100.130 mg/L (99.666 mg/L without algae) after 0 h; 99.695 mg/L (100.945 mg/L without algae) after 72 h
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
- Initial cells density: approximately 5000 cells per millilitre
- No. of vessels per concentration (replicates): 6; additionally test concentration without algae. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the limit test concentration without algae was run in parallel to the test concentration.
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium of OECD TG 201 was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item; see attachment 4)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (attachment 4).

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity and quality: 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.
- Determination of cell concentrations: Cell density measurements were made using a Cellcounter Z2, Beckman Coulter.

TEST CONCENTRATIONS
- Range finding study: A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test.
- Test concentrations: 0.1, 1, 10 and 100 mg/L.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
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
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:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to EU method C.3, which is equivalent to OECD guideline 201, on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards algae.
No toxic effects against algae were observed at a limit test concentration of 100 mg/L at the limit of water solubility under exposure conditions. EC10, EC50 and LOEC are > 100 mg/L and NOEC ≥ 100 mg/L (all after 72 h based on growth rate).
Based on the obtained results, the substance does not need to be classified as hazardous to the aquatic environment according to the Regulation (EC) No. 1272/2008.
Executive summary:

A GLP-study was performed to assess the adverse effects of Hydroxytoluic acid on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

 The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of Hydroxytoluic acid dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and a folded filter.

 The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to STUDENT-t test for Homogeneous Variances. The following values were determined: 

Results [mg/L]:

 ErC 50* (0-72 h):           > 100

 ErC 10* (0-72 h):           > 100

 NOEC [r]* (tα 0.05):          ≥100

 LOEC [r]* (tα 0.05):           > 100

 

No toxic effects against algae were observed at a limit test concentration of 100 mg/L at the limit of water solubility under exposure conditions.

The results are expressed in terms of nominal concentrations. Effective concentration correspond to 100.1 % of nominal values at 0 hours and to 99.7 %of nominal values at72 hours.