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:
08 Feb 2018 - 01 Apr 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)
Version / remarks:
2011
Qualifier:
according to
Guideline:
other: OECD Guideline 23 (Guidance document on aquatic toxicity testing of difficult substances and mixtures)
Version / remarks:
2000
GLP compliance:
yes
Remarks:
Quality Assurance Statement included (11 Apr 2019)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Remarks:
GC-MS
Details on sampling:
- Concentrations: All concentrations and the control were analytically measured at the beginning and at termination of the experiment.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Because of the poorly solubility in water of the test item an extraction has been performed to extract the soluble components of the test item in water, according to OECD Guidance N. 23. The test item has been prepared at 100 mg/L and 31.25 mg/L in culture medium. The solutions have been left in contact in medium for 72 h, at 10 rpm and room temperature. After this contact time the solutions at 9.77, 3.05 and 0.95 mg/L were prepared under stirring from the obtained 31.25 mg/L solution.
- Controls: culture medium
- Evidence of undissolved material: No, after stirring the solutions appeared clear.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source: Laboratory culture (original source: Sammlung von Algenkulturen der Universität Göttingen).
- Age of inoculum (at test initiation): algal suspension, prepared from the stock algal culture was breeding 3 days before the beginning of the test.
- Method of cultivation: in culture medium in 250 mL Erlenmeyer flasks, 23°C, lighting > 4300 lux

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
23 - 23.2 °C
pH:
7.44 - 7.91
Nominal and measured concentrations:
Nominal: 100, 31.25, 9.77, 3.05, 0.95 and 0 (control) mg/L
Measured initial: 58.33, 26.01, 8.15, 2.97, 0.94 and 0 (control) mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Initial cells density: 10E-04 cells/mL
- Control end cells density: between 2.2 E+06 and 2.5 E+06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the experiment. Temperature was recorded every 24 h.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7927 - 7967 Lx

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell density measured every 24 h by Burker chamber. At the end of the test the quality of algae appearance was observed under the microscope.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 and 0 (control) mg/L
- Results used to determine the conditions for the definitive study: Yes, but no details given in report.
Reference substance (positive control):
not specified

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 58.33 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: test item stable during test
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.41 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: test item concentration stable during test
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: normal and healthy appearance of algae at the end of the test was observed microscopically
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Any other information on results incl. tables

ANALYTICAL RESULTS:

Table 1: Recovery of test item after 0 and 72 h.

Nominal concentration T = 0

[mg/L]

Measured concentration T= 0

[mg/L]

Measured concentration T= 72 h

[mg/L]

% of recovery at 72 h compared to 0 h

100

58.33

51.8

88.76

31.25

26.01

24.85

95.54

9.77

8.15

7.65

93.92

3.05

2.97

2.61

87.87

0.95

0.94

0.77

81.53

The final EC50 value has been reported without statistical analysis since the highest tested concentration has shown an inhibition % lower than 50%. The substance was however tested up to the max of its water solubility.

BIOLOGICAL RESULTS

Table 2: Algae density (cells/mL) in treatments.

Days

1

2

3

Treated Group 58.33 mg/L

 

 

 

0

1 E+04

1 E+04

1 E+04

1

2.3 E+04

2.5 E+04

2.3 E+04

2

6.7 E+04

8.2 E+04

7 E+04

3

2.2E+05

2.2 E+05

2.3 E+05

Treated Group 26.01 mg/L

 

 

 

0

1 E+04

1 E+04

1 E+04

1

3.2 E+04

4 E+04

3.5 E+04

2

1.3 E+05

1.4 E+05

1.3 E+05

3

3 E+05

2.9 E+05

3.3 E+05

Treated Group 8.15 mg/L

 

 

 

0

1 E+04

1 E+04

1 E+04

1

4.5 E+04

4.3 E+04

4.8 E+04

2

3.3 E+05

3.9 E+05

3.8 E+05

3

1.2 E+06

1.2 E+06

1.2 E+06

Treated Group 2.97 mg/L

 

 

 

0

1 E+04

1 E+04

1 E+04

1

6.5 E+04

5.7 E+04

6.2 E+04

2

6 E+05

5.7 E+05

6.2 E+05

3

2.2 E+06

2 E+06

2.2 E+06

Treated Group 0.94 mg/L

 

 

 

0

1 E+04

1 E+04

1 E+04

1

6.7 E+04

5.8 E+04

6.2 E+04

2 E+05

6.4 E+05

6.2 E+05

5.8 E+05

3 E+06

2.3 E+06

2.2 E+06

2.4 E+06

Table 3: Algae density (cells/mL) in controls.

Days/Controls

1

2

3

4

5

6

0

1 E+04

1 E+04

1 E+04

1 E+04

1 E+04

1 E+04

1

7 E+04

6 E+04

6 E+04

5.3 E+04

6.5 E+04

6.3 E+04

2

6.5 E+05

6.4 E+05

6.2 E+05

5.9 E+05

6.3 E+05

6 E+05

3

2.4 E+06

2.5 E+06

2.2 E+06

2.3 E+06

2.4 E+06

2.3 E+06

Table 4: % inhibition of growth rate.

Parameter

Treated

58.33 mg/L

26.01 mg/L

8.15 mg/L

2.97 mg/L

0.94 mg/L

%Ir

43.08

37.30

12.36

1.7

0.28

VALIDITY CRITERIA:

Table 5: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

>a factor of 16

yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

27.19

yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

0.93

yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.