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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:
16 Oct - 19 Oct 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
interministerial group of chemical products (GIPC)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dodecanoic acid, ester with 1,2-propanediol
EC Number:
253-462-2
EC Name:
Dodecanoic acid, ester with 1,2-propanediol
Cas Number:
37321-62-3
Molecular formula:
C15H30O3 C27H52O4
IUPAC Name:
Dodecanoic acid, ester with 1,2-propanediol

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.95, 3.05, 9.76, 31.25 and 100 mg/L at 0 and 72 hours.
- Sampling method: Test item solutions and control were sampled and immediately analysed. Diester: 2 mL of sample were pipetted into a 15 mL conical bottom glass tube and 2 mL of ethyl acetate were added. Samples were vortexed and layers were allowed to separate for one minute. 1 mL of the upper layer was pipetted into a GCMS vial. Monoester: 2 mL of sample were pipetted into a 15 mL conical bottom glass tube and 2 mL of hexane were added. Samples were vortexed and centrifuged for 2 min at 1500 rpm. 1 mL of the upper layer was pipetted into a GCMS vial and 50 μL of derivative agent BSTFA + 1 % TMCS were added.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were prepared individually by water-accommodated fractions (WAF) approach according to OECD monograph No. 23. WAFs were prepared individually. Measured quantities of test item were added directly to test medium and mixed for 24 hours to achieve an equilibrated concentration of dissolved and dispersed or emulsified components in the aqueous phase. The test solutions have not been filtered. The test solutions used were therefore emulsions of the test element.
- Controls: yes (test medium)

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): The algal stock culture was provided by the CCAP (Culture Collection of Algae and Protozoa), reference CCAP 1405/1.
- Method of cultivation: The Pseudokirchneriella subcapitata stock culture was maintained on sloped agar tubes at 4°C and regularly transferred to fresh medium at the laboratory. A pre-culture was prepared from the stock culture and maintained in an exponential growth by successive transfers every 3 to 4 days.

ACCLIMATION
- Culturing media and conditions (same as test or not): OECD medium

Study design

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

Test conditions

Test temperature:
21.63 - 22.69 °C
pH:
7.66 - 8.81
Nominal and measured concentrations:
nominal (loading rates): 0.95, 3.05, 9.76, 31.25 and 100 mg/L
measured: see below table 2 under "Any other information on results
Details on test conditions:
TEST SYSTEM
- Test vessel: glass Erlenmeyer flasks of 250 mL
- Type (delete if not applicable): closed with air-permeable stoppers made of polyether foam
- Fill volume: 80 mL
- Initial cells density: 5000 cells per mililiters
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- other: The flasks were repositioned each day to minimize differences in test conditions.

GROWTH MEDIUM
- Standard medium used: OECD medium in accordance with OECD 201

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: pH was recorded at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 7700 - 85000 lux
- other: Algae were maintained suspended under continuous orbital stirring.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell biomass was measured every day in each flask using an electronic particle counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.02, 0.2, 2 and 20 mg/L
- Results used to determine the conditions for the definitive study: Growth rate inhibition: 7.59% Inhibition at 0.02 mg/L, 2.91% Inhibition at 0.2 mg/L, 3.65% at 2 mg/L and 56.01% at 20 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
9.76 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% CL 27.93 - 33.29 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% CL 11.86 – 26.98 mg/L
Details on results:
- Observation of abnormalities (for algal test): No abnormal appearance of the algae at the start and at the end of the test for all concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 (72h) = 1.07 mg/L (95% CL 0.94 – 1.2); starting date 18 May 2018
Reported statistics and error estimates:
EC10 (Effect Concentration for 10% effect), EC50 and confidence limits were obtained by calculation according to Regtox® Macro (“Vindimian”), using the Hill model.
The statistical evaluation (NOEC - No Observed Effect Concentration - and the LOEC - Lowest Observed Effect Concentration - determination) was conducted with R software.

Any other information on results incl. tables

The components were not detectable at low concentrations and the evaluation of the endpoints were based on nominal loading rates.

Table 1: 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.

The growth multiplication factor in the control within the 72-hour test period was 288.

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%

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1- 2 and 2-3) in the control was 11.2%.

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%.

The coefficient of variation of the average specific growth rates in the control during the whole test period was less than 6.16%.

yes

 

Table 2: Results for the controls after 72h

 

 

Number of cells at 0 h

Measure at 24 h (cells / mL)

Measure at 48 h (cells / mL)

Measure at 72 h (cells / mL)

Specific growth rate 0 h

– 24 h

Specific growth rate 24 h – 48 h

Specific growth rate 48 h – 72 h

Growth rate (day-1) after 72h

CV

growth 72h (%)

Yield (cells / mL) after 72h

CV

yield 72h (%)

 

 

 

Control

5000

33112

210200

1705000

1.890

1.848

2.093

1.944

 

 

 

6.2

1700000

 

 

 

28.7

5000

23328

129500

1133000

1.540

1.714

2.169

1.808

1128000

5000

23432

137700

738800

1.545

1.771

1.680

1.665

733800

5000

26112

243400

1629000

1.653

2.232

1.901

1.929

1624000

5000

33032

247400

1702000

1.888

2.014

1.929

1.943

1697000

5000

24616

179900

1743000

1.594

1.989

2.271

1.951

1738000

Mean

5000

27272

191350

1441800

1.685

1.928

2.007

1.873

1436800

 

Table 3: Results for all test concentrations after 72h

Nominal loading rates (mg test item/L after 72h)

 

 

Number of cells at 0 h

 

 

Measure at 24 h (cells / mL)

 

 

Measure at 48 h (cells / mL)

 

 

Measure at 72 h (cells / mL)

 

Growth rate (day-1) after 72h

 

 

CV

growth 72h (%)

Growth rate inhibition (%)

compared to control after 72h

 

 

Yield (cells / mL) after 72h

 

 

 

CV yield 72h (%)

 

Yield inhibition (%)

compared to control after 72h

 

0.950

5000

33144

344800

1453000

1.891

 

9.1

-0.92

1448000

 

50.0

-0.78

5000

29952

137600

2412000

2.060

-9.94

2407000

-67.53

5000

34920

142800

856800

1.715

8.48

851800

40.72

Mean

5000

32672

208400

1573933

1.888

-0.79

1568933

-9.20

 

3.050

5000

22968

391500

865300

1.718

 

9.2

8.30

860300

 

46.8

40.12

5000

36248

265800

2421000

2.061

-10.01

2416000

-68.15

5000

25264

253600

1719000

1.947

-3.91

1714000

-19.29

Mean

5000

28160

303633

1668433

1.908

-1.87

1663433

-15.77

 

9.760

5000

22488

133300

1123000

1.805

 

3.6

3.66

1118000

 

18.0

22.19

5000

17272

128500

791500

1.688

9.89

786500

45.26

5000

16224

140400

1073000

1.790

4.47

1068000

25.67

Mean

5000

18661

134067

995833

1.761

6.01

990833

31.04

 

31.700

5000

5888

1280

127100

1.079

 

13.9

42.43

122100

 

44.4

91.50

5000

14913

1240

62480

0.842

55.07

57480

96.00

5000

0*

1280

67760

0.869

53.62

62760

95.63

Mean

5000

6934

1267

85780

0.930

50.37

80780

94.38

 

100.420

5000

0*

1760

120

0.000

 

-

100.00

0

 

-

100.00

5000

0*

17400

160

0.000

100.00

0

100.00

5000

0*

1400

80

0.000

100.00

0

100.00

Mean

5000

0

6853

120

0.000

100.00

0

100.00

 

Table 4: Analytical results

Treatment

Control

Test substance

ID

C06

C07

C08

C09

C10

C11

Actual loading rate [mg test item.L-1]

0

0.95

3.05

9.76

31.7

100.42

0h  analyzed concentrati on

[mg monoester.L-1]

< LOQ

< LOQ

< LOQ

0.32

3.66

16.17

[mg diester.L-1]

< LOQ

< LOQ

< LOQ

1.95

8.91

25.37

72h analyzed concentrati on [mg test item.L-1]

[mg monoester.L-1]

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

0.26

[mg diester.L-1]

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

4.81

Limit of quantification (LOQ) =0.1 mg.L-1 for monoester, and 1 mg.L-1 for diester.

Applicant's summary and conclusion

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