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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:
2018-01-10 to 2018-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted on March 23, 2006; Annex 5 corrected: July 28, 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Remarks:
The test item concentration in the reconstituted water had low water solubility (<0.011 mg/L), thus, the compound cannot be detected with standard analytical methods.
Vehicle:
yes
Remarks:
reconstituted water
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study. The pH value was not adjusted.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: 61.81 SAG
- Source: „Pflanzenphysiologisches Institut der Universität Göttingen“, Germany.
- Method of cultivation: The permanent algae cultures, the pre-culture and the algae cultures of the study were cultivated in an air-conditioned room with a water temperature of 21.0 – 24.0 °C, controlled at ± 2 °C. For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers. The cultures were shaken continuously at about 120 rpm and lit between 4440 and 8880 Lux. Fluorescent tubes installed above the flasks served for lighting.

ACCLIMATION
- Acclimation period: 72-h
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
-
Hardness:
24 mg/L as CaCO3 (calculated)
Test temperature:
24.4 to 25.3 °C
pH:
7.90 to 8.92
Nominal and measured concentrations:
Nominal concentrations: 100.0 mg/L
No measured concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glass flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10 000 cells per mL
- Control end cells density: 1 756 338 cells per mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD TG 201
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The mean light intensities were 7544 Lux ± 2.8 % and 7298 Lux ± 2.6 % prior to and at the end of the exposure period, respectively.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Nominal test concentration: 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: >0.011 mg/L (limit of water solubility)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: >0.011 mg/L (limit of water solubility)
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
Remarks on result:
other: >0.011 mg/L ( (limit of water solubility)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: >0.011 mg/L ( (limit of water solubility))
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: >0.011 mg/L (limit of water solubility)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: >0.011 mg/L (limit of water solubility)
Details on results:
No significant effect on growth of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility.

The EC values exceeded the water solubility of <0.011 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
The test item (Potassium dichromate) showed 72h EC50 of 0.91 mg/L for growth rate and 0.48 mg/L for yield. The EC50 for growth rate and yield obtained in this study are in the range of ± two standard deviation of the current historical means.

Therefore, this study confirms the sensitivity and reliability of the test system used in the test facility.

Table 1: Algal Growth Measurement - Algal biomass (cell number) after 24, 48 and 72 hour exposure

Nominal concentration [mg/L]

Biomass of algae (Cell number per mL*)

Control

 

0 hours

24 hours

48 hours

72 hours

1.0000

6.5340

30.5460

167.6043

1.0000

6.1713

38.6757

210.6063

1.0000

5.2920

28.4724

139.7943

1.0000

5.3910

34.5627

171.3303

1.0000

5.5746

31.7034

151.6023

1.0000

5.8896

38.3994

212.8653

Mean

1.0000

5.8087

33.7266

175.6338

SD

0.00000

0.48173

4.21530

30.18024

CV

0.0

8.3

12.5

17.2

100.0

 

0 hours

24 hours

48 hours

72 hours

1.0000

5.0094

30.0780

176.1480

1.0000

5.1507

31.0743

185.2560

1.0000

5.9517

36.5220

192.9510

1.0000

5.6520

34.6680

187.5780

1.0000

5.2884

31.7376

182.8710

1.0000

5.5557

34.0740

186.9570

Mean

1.0000

5.4347

33.0257

185.2935

SD

0.00000

0.34988

2.45668

5.58809

CV

0.0

6.4

7.4

3.0

*Cell number divided by factor 10000

 

Table 2: Yield after 24, 48 and 72 hour exposure

Nominal concentration [mg/L]

Yield

Control

 

24 hours

48 hours

72 hours

5.5340

29.5460

166.6043

5.1713

37.6757

209.6063

4.2920

27.4724

138.7943

4.3910

33.5627

170.3303

4.5746

30.7034

150.6023

4.8896

37.3994

211.8653

Mean

4.8087

32.7266

174.6338

SD

0.48173

4.21530

30.18024

CV

10.0

12.9

17.3

100.0

 

24 hours

48 hours

72 hours

4.0094

29.0780

175.1480

4.1507

30.0743

184.2560

4.9517

35.5220

191.9510

4.6520

33.6680

186.5780

4.2884

30.7376

181.8710

4.5557

33.0740

185.9570

Mean

4.4346

32.0257

184.2935

SD

0.34988

2.45668

5.58809

CV

7.9

7.7

3.0

 

Table 3: Mean specific growth rate after 24, 48 and 72 hour exposure

 

Nominal concentration [mg/L]

Mean specific growth rate

Control

 

24 hours

48 hours

72 hours

1.877

1.710

1.707

1.820

1.828

1.783

1.666

1.674

1.647

1.685

1.771

1.715

1.718

1.728

1.674

1.773

1.824

1.787

Mean

1.757

1.756

1.719

SD

0.0820

0.0625

0.0569

CV

4.7

3.6

3.3

100.0

 

24 hours

48 hours

72 hours

1.611

1.702

1.724

1.639

1.718

1.741

1.784

1.799

1.754

1.732

1.773

1.745

1.666

1.729

1.736

1.715

1.764

1.744

Mean

1.691

1.747

1.741

SD

0.0641

0.0371

0.0101

CV

3.8

2.1

0.6

 

Table 4: EC-Values

Parameter (0-72h)

Growth rate

Yield

EC10

95% confidence interval

>0.011 mg/L (nominal >100 mg/L) n.d.

>0.011 mg/L (nominal >100 mg/L) n.d.

EC20

95% confidence interval

>0.011 mg/L (nominal >100 mg/L) n.d.

>0.011 mg/L (nominal >100 mg/L) n.d.

EC50

95% confidence interval

>0.011 mg/L (nominal >100 mg/L) n.d.

>0.011 mg/L (nominal >100 mg/L) n.d.

 

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.
The 72h EC50, EC20 and EC10 for growth rate and yield were >0.011 mg/L (nominal >100 mg/L) and thus, could not be determined in this study.


Executive summary:

The toxicity of the test item on the growth and growth rate of the green algae of the unicellular green alga,Pseudokirchneriella subcapitataduring exposure period of 72 hours was estimated in a method according to OECD 201 under GLP. For this purpose, exponentially growing cultures ofPseudokirchneriella subcapitatawere exposed to a nominal test item concentration of 100 mg/L. The study comprised of one control group and one test item group with six replicates each. The initial algae cell density in each replicate was 10 000 cells/mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. The test item concentration in the reconstituted water was not quantified at the start and the end of this study. Because of the low water solubility (<0.011 mg/L), the test item cannot be detected with standard analytical methods and the development of an analytical method with a sufficiently low detection and quantification limit is complex. Due to the absence of any adverse effects at the saturation concentration, the study was performed without analytical concentration verification. No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50, EC20 and EC10for growth rate and yield were >0.011 mg/L (nominal >100 mg/L) and thus, could not be determined in this study

Description of key information

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.

The 72h EC50, EC20 and EC10for growth rate and yield were >0.011 mg/L (nominal >100 mg/L) and thus, could not be determined in this study. (reference 6.1.5-1).

Key value for chemical safety assessment

Additional information

The toxicity of the test item on the growth and growth rate of the green algae of the unicellular green alga,Pseudokirchneriella subcapitataduring exposure period of 72 hours was estimated in a method according to OECD 201 under GLP. For this purpose, exponentially growing cultures ofPseudokirchneriella subcapitatawere exposed to a nominal test item concentration of 100 mg/L. The study comprised of one control group and one test item group with six replicates each. The initial algae cell density in each replicate was 10 000 cells/mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. The test item concentration in the reconstituted water was not quantified at the start and the end of this study. Because of the low water solubility (<0.011 mg/L), the test item cannot be detected with standard analytical methods and the development of an analytical method with a sufficiently low detection and quantification limit is complex. Due to the absence of any adverse effects at the saturation concentration, the study was performed without analytical concentration verification. No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50, EC20 and EC10for growth rate and yield were >0.011 mg/L (nominal >100 mg/L) and thus, could not be determined in this study