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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:
July 15, 2016 - November 4, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
Designation: Art. 201386
Synonym: B-2O-O5
Batch: EF13004186
Purity (HPLC):100.0%
Appearance: White solid
Water solubility: <0.03 mg/L (OECD 105)
Stability under
test conditions: Considered stable (non-degradable, not volatile)
Date of expiry: April 30, 2018
Storage: Tightly closed, dark at room temperature (15 to 25°C)


PREPARATION OF THE TEST MATERIAL
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 further 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study.

Sampling and analysis

Analytical monitoring:
no
Remarks:
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 (<0.03 mg/L) the compound cannot be detected with standard analytical methods.

Test solutions

Vehicle:
yes
Details on test solutions:
Vehicle:

Nutrient (mg/L)
NH4Cl 15.0
MgCl2 * 6H2O 12.0
CaCl2 * 2H2O 18.0
MgSO4 * 7H2O 15.0
KH2PO4 1.60

FeCl3 * 6H2O 0.0640
Na2EDTA * 2H2O 0.100

H3BO3 0.185
MnCl2 * 4H2O 0.415
ZnCl2 0.00300
CoCl2 * 6H2O 0.00150
CuCl2 * 2H2O 0.00001
Na2MoO4 * 2H2O 0.00700

NaHCO3 50.0

- pH: 7.86
- Total hardness: 24 mg/L as CaCO3 (calculated)

Preparation of the Test Item:
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 further 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): The test system used was Pseudokirchneriella subcapitata, strain 61.81 SAG (Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany). This strain was further cultivated in the laboratories of Non-Clinical Safety.
- 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 (Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany). The cultures were shaken continuously at about 120 rpm (Universalschüttler SM25, Edmund Bühler GmbH, Hechingen, Germany) and lit between 4440 and 8880 Lux. Fluorescent tubes (Lumilux T5 nws FLH1 HO 80W/840, Osram GmbH, München, Germany) installed above the flasks served for lighting.


ACCLIMATION
- Acclimation period: 72h
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
-

Test conditions

Hardness:
24 mg/L as CaCO3 (calculated)
Test temperature:
23.2 - 24.0°C
pH:
7.86 - 10.35

The pH in the control group did increase more than 1.5 units during the test. This did not influence the outcome of the study.
Nominal and measured concentrations:
Nominal concentrations: 100.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glas flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10 000 cells per ml
- Control end cells density: 3 680 247 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 light intensity measured immediately before the start of the study was 8093 Lux ± 4.7% and 8055 Lux ± 3.9% at the end of the study. Thus, the light intensity was in the anticipated range of 4440 – 8880 Lux within ± 15% from the average.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Nominal test concentration: 100 mg/L
Reference substance (positive control):
no
Remarks:
The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control (Gado, 2016).

Results and discussion

Effect concentrationsopen allclose all
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: EC10 >0.03 mg/L
Key result
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: EC20 >0.03 mg/L
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: EC50 >0.03 mg/L
Key result
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: EC10 >0.03 mg/L
Key result
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: EC20 >0.03 mg/L
Key result
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: EC50 >0.03 mg/L
Details on results:
The EC values exceeded the water solubility of 0.03 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 accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control (Gado, 2016).

Under the conditions of the present study, the test item Art. 104864 (Potassium dichromate) showed 72h EC50 of 1.03 mg/L for growth rate and 0.53 mg/L for yield. The results obtained in this study are similar to results of a ring test mentioned in DIN EN ISO 8692.

Any other information on results incl. tables

Objective

The objective of the present study was to determine the effect of Art. 201386 (B-2O-O5) on growth rate and yield of the unicellular green alga Pseudokirchneriella subcapitata.

Study Design

The study comprised of one control group and one test item group with six replicates each. The nominal test item concentration of 100 mg/L was dissolved in 100 mL reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group.

Results

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 (<0.03 mg/L), the compound cannot be detected with standard analytical methods and the development of an analytical method with a sufficiently low detection and quantification limit is complex. The test item is considered stable in water.

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 of the algae was observed at a nominal concentration of 100 mg/L.

For the test item Art. 201386 (B-2O-O5) the following EC values were determined:

 Parameter (0 -72h) Growth Rate   Yield

EC10 [mg/L]

95% confidence interval

>0.03 mg/L (nominal >100 mg/L)

n.d. 

 >0.03 mg/L (nominal >100 mg/L)

n.d.

EC20 [mg/L]

95% confidence interval

 >0.03 mg/L (nominal >100 mg/L)

n.d.

 >0.03 mg/L (nominal >100 mg/L)

n.d.

EC50 [mg/L]

95% confidence interval

 >0.03 mg/L (nominal >100 mg/L)

n.d.

 >0.03 mg/L (nominal >100 mg/L)

n.d. 

Conclusion

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of Art. 201386 (B-2O-O5) revealed no aquatic toxicity in the test system.

The 72h EC50for growth rate and yield were >0.03 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of Art. 201386 (B-2O-O5) revealed no aquatic toxicity in the test system.

The 72h EC50 for growth rate and yield were >0.03 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of Art. 201386 (B-2O-O5) revealed no aquatic toxicity in the test system.The 72h EC50 for growth rate and yield were >0.03 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.