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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:
09 Feb - 15 Feb 2018
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2-bis[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate
EC Number:
238-430-8
EC Name:
2,2-bis[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate
Cas Number:
14450-05-6
Molecular formula:
C41H76O8
IUPAC Name:
2,2-bis[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples for analysis were taken from all test concentrations and the control at test start, after 24 h and at test end. 6.0 mL from the approximate centre of the test vessels were taken for each sample. In addition, the glass wool containing the undissolved residue was kept for possible analysis.
- Sample storage conditions before analysis: No storage; samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for an overnight period. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E+04 cells/mL.
- Eluate: no
- Differential loading: yes
- Controls: yes, blank control and reference control
- Evidence of undissolved material: No, all test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater green algae
- Source: in-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C and a light intensity of 60 to 120 µE/m2/s (wavelength range of 400 to 700 nm). The stock culture medium M1 was prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis).

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: The pre-culture medium M2 was prepared according to the OECD 201 Guideline (same as test medium).
- Any deformed or abnormal cells observed: no abnormalities observed

Study design

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

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22 - 23 °C
pH:
Control: 8.2 - 8.3
Treatment (100 mg/L): 8.0 - 8.2
Nominal and measured concentrations:
nominal: control, 1.0, 10 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass vessels, containing 50 mL of test solution
- Type: capped vessels
- Initial cells density: 10E+04 cells/mL
- Control end cells density: 139.4 x 10E+04 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured continuously in a temperature control vessel. The pH was measured at the beginning and at the end of the test, for the control and limit concentration.

OTHER TEST CONDITIONS
- Photoperiod: continuous light
- Light intensity and quality: TLD-lamps with a light intensity within the range of 86 to 87 µE.m-2s-1

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions. Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using fewer concentrations than requested by guideline: combined limit/range-finding test
- Range finding study: The main test is a combined limit/range-finding test.
- Test concentrations: 1.0, 10 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate; performed a maximum of 3 months before or after the start of the test item project

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Details on results:
In the samples taken at the start of the test, small responses were measured which could not be quantified with the method available. These responses were considered to be not test item related since a comparable response was measured in the control. After 24 hours of exposure, no response was measured in the control and at any of the test concentrations. Therefore, samples taken at the end of the test were not analysed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.86 mg/L (95% CI: 0.84 - 0.88 mg/L)
Reported statistics and error estimates:
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

For determination of the NOEL, the approach recommended for the determination of the NOEC in the OECD guideline 201 was used. An effect was considered to be significant if statistical analysis of the data obtained for the limit concentration compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Two-Sample t-Test Procedure, α=0.05, one-sided, smaller). No EL10 and EL50-values could be calculated because the recorded effects were below 10%.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1: Growth Rate And Percentage Inhibition For The Total Test Period

Test item1
Loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.645

0.0235

6

1.0

1.662

0.0281

3

-1.0

10

1.676

0.0035

3

-1.9

100

1.645

0.0262

6

0.0010

1– 2,2-bis[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate

Table 2: Yield And Percentage Inhibition For The Total Test Period

Test item1
Loading rate (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

138.4

9.76

6

1.0

145.7

12.26

3

-5.2

10

151.6

1.60

3

-9.5

100

138.5

11.55

6

-0.059

1– 2,2-bis[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate

Table 3: Individual Cell Densities (x104cells/mL)

Time

Replicate

2,2-bis[[(1-oxononyl)oxy]methyl]propane-1,3-diyl dinonan-1-oate Loading rate (mg/L)

Control

1.0

10

100

0 h

1

2

3

4

5

6

1.000

1.000

1.000

1.000

 

2

1.000

1.000

1.000

1.000

 

3

1.000

1.000

1.000

1.000

 

4

1.000

 

 

1.000

 

5

1.000

 

 

1.000

 

6

1.000

 

 

1.000

 

 

 

 

 

n:

 

6

3

3

6

Mean:

 

1.0

1.0

1.0

1.0

Std.Dev.:

 

0.0

0.0

0.0

0.0

CV:

 

0.0

0.0

0.0

0.0

 

 

 

 

 

24 h

1

2

3

4

5

6

6.300

n.d.

n.d.

6.249

 

2

6.568

n.d.

n.d.

6.577

 

3

7.009

n.d.

n.d.

6.751

 

4

6.857

 

 

6.331

 

5

6.215

 

 

6.855

 

6

6.307

 

 

6.076

 

 

 

 

 

n:

 

6

3

3

6

Mean:

 

6.5

n.a.

n.a.

6.5

Std.Dev.:

 

0.3

n.a.

n.a.

0.3

CV:

 

5.0

n.a.

n.a.

4.7

 

 

 

 

 

48 h

1

2

3

4

5

6

28.985

n.d.

n.d.

28.703

 

2

29.779

n.d.

n.d.

28.054

 

3

33.162

n.d.

n.d.

30.098

 

4

30.556

 

 

28.107

 

5

30.221

 

 

33.664

 

6

29.292

 

 

28.298

 

 

 

 

 

n:

 

6

3

3

6

Mean:

 

30.3

n.a.

n.a.

29.5

Std.Dev.:

 

1.5

n.a.

n.a.

2.2

CV:

 

5.0

n.a.

n.a.

7.4

 

 

 

 

 

72 h

1

2

3

4

5

6

126.208

133.741

151.075

135.473

 

2

131.277

158.122

152.462

132.230

 

3

151.149

148.141

154.274

138.426

 

4

137.157

 

 

130.891

 

5

142.328

 

 

162.306

 

6

148.575

 

 

137.855

 

 

 

 

 

n:

 

6

3

3

6

Mean:

 

139.4

146.7

152.6

139.5

Std.Dev.:

 

9.8

12.3

1.6

11.6

CV:

 

7.0

8.4

1.1

8.3

n.d. – not determined, n.a. – not applicable.

Table 4: Validity criteria

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.

 139

 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%

 12%

 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%.

 1.4%

 yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
No effect on aquatic algae was observed up to the limit of water solubility of the registered substance CAS 104450-05-6.