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Ecotoxicological information

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:
2015-07-27 to 2015-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light, in the tightly closed original container.
- Stability under test conditions: Assumed to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: 19.0 g/L (pH 4.4, 20 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of 130 mg/L was freshly prepared with dilution water. Manual agitation until the solution was clear.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The limit concentration and the control were analytically verified via GC-FID after 0 hours (start of exposure) and 72 hours (end of the exposure).
- Sampling method: Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours separate replicates were prepared with algae at the beginning of the exposure and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at room temperature before and after preparation.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 130 mg/L was freshly prepared with dilution water. For preparation of the test concentration the density was taken into account. An appropriate volume of the test item was pipetted directly into measuring flask, containing the respective volume of the dilution water. After dispersion treatment the prepared test solution was transferred to the test vessels which were tightly closed, without headspace. Manual agitation was done until the solution was clear.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test item replicates.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green freshwater algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35-70 µE x m-2 x s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period: 72 hours.
- Culturing media and conditions (same as test or not): The culturing conditions were not the same as the test conditions (the light intensity in the test was higher: 60-120 µE x m-2 x s-1).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L (dilution water)
Test temperature:
22°C (mean)
pH:
after 72 h: 8.45 (concentration), 8.59 (control)
Nominal and measured concentrations:
at 0 h: 130 mg/L (nominal); 125 mg/L (measured)
at 72 h: 130 mg/L (nominal); 115 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks, volume: 119 mL, with aluminium tops with PTFE seals.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Filled without headspace.
- Aeration: No.
- Renewal rate of test solution (frequency/flow rate): No.
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL; Current: 6153 cells/mL.
- Control end cells density: 624869 cells /mL (72 h).
- No. of vessels per concentration (replicates): 6 replicates.
- No. of vessels per control (replicates): 6 replicates.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD 201.
- Culture medium different from test medium: Yes

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 24 hours/day light.
- Light intensity and quality: 60 - 120 µE*m^-2*s^-1 (nominal).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Via Chlorophyll-a-fluorescence.
- Other: Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Test concentrations: nominal: 0 and 130 mg/L; measured: < LOQ and 115 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 130 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.:
> 130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 130 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- The measured concentration of Ethyl propionate in the limit concentration at the start of the exposure was 96 % and at the end of the exposure 88 % of the of the nominal test item concentration. Therefore, all effect levels are given based on the nominal test item concentration.
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the reference item toxicity is in the valid range following test facility SOPs.
- ErC50 with headspace: 0.613 mg/L; without headspace: 0.774 mg/L

Table 1: Cell Densities

Nominal Test Item Concentration [mg/L]

Mean Cell Density [cells/mL]

0 hours

24 hours

48 hours

72 hours

130

6153

21721

182685

540244

Control

6153

24138

182050

624869

 

Table 2 Evaluation after 72 hours

Nominal Test Item Concentration [mg/L]

Growth rate (d-1)

Growth rate inhibition (%)

Yield (cells/mL)

Yield inhibition (%)

130

(+)* 1.49

3

(+) 534091

14

Control

1.54

 

618716

 

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-)

* = not biologically significant

Table 3: Measured Concentrations and Percent of Nominal Concentration of Ethyl propionate

Nominal test item concentration [mg/L]

Start of exposure (0 hours)

End of exposure (72 hours)

Meas. conc. [mg/L]

(%)

Meas. conc. [mg/L]

(%)

130

125

96

115

88

Control

< LOQ

< LOQ

Meas. conc. = Measured concentration of the test item, enrichment and dilution factor taken into account

LOQ = Limit of quantification of the analytical method (10.0 mg test item/L)

(%) = Percent of the nominal test item concentration

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test substance to the green freshwater algae Pseudokirchneriella subcapitata was determined according to OECD guideline 201 and GLP. The results are as follows:
EC50 (72 h) > 130 mg/L for both, inhibition of growth rate and yield

ErC10 (72 h) > 130 mg/L for growth rate inhibition
EyC10 (72 h) < 130 mg/L for inhibition of yield
NOEC (72 h) = 130 mg/L for inhibition of growth rate
NOEC (72 h) < 130 mg/L for yield inhibition
(based on nominal concentrations).

Description of key information

EC50 (72 h) > 130 mg/L for both, inhibition of growth rate and yield

ErC10 (72 h) > 130 mg/L for growth rate inhibition

EyC10 (72 h) < 130 mg/L for inhibition of yield

NOEC (72 h) = 130 mg/L for inhibition of growth rate

NOEC (72 h) < 130 mg/L for yield inhibition

(based on nominal concentrations).

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
130 mg/L
EC10, LC10 or NOEC for freshwater algae:
130 mg/L

Additional information

The toxicity of the test substance to the green freshwater algae Pseudokirchneriella subcapitata was determined according to OECD guideline 201 and GLP. The Algae were exposed to the test substance in a limit test with the tested concentration being 130 mg/L. The limit concentration and the control were analytically verified via GC-FID after 0 and 72 hours. The EC50 (72h) was > 130 mg/L for both, inhibition of growth rate and yield. The ErC10 (72h) was >130 mg/L for growth rate inhibition and EyC10 < 130 mg/L for inhibition of yield. The NOEC for inhibition of growth rate after 72 hours was 130 mg/L and for yield after 72 hours < 130 mg/L. The results are based on nominal concentrations.