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:
Experimental starting date: 2015-05-19; experimental completion date: 2015-05-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
resp. EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
1. the temperature range for algal growth was 20.4 - 22.6°C instead of 21 - 24 °C; 2. the test item was stored at 15.6 - 26.8°C instead of 15 - 20°C; Both deviations can be seen as uncritical for the outcome of the study
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Kaiser-Friedrich-Straße 7; D-55116 Mainz; Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: viscous liquid
Details on test material:
Stabilized EMIM BF4:
Appearance: lightly yellow clear viscous liquid
Purity: 93.36% (EMIM+, HPLC); 86.68% (BF4-, titration); 4.89% (Stabilizer, HPLC)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
stock solution containing 99.9 mg/L test item

Test solutions

Details on test solutions:
Stock solution I:
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL

Stock solution II:
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL

Stock solution III:
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL

Stock solution IV:
NaHCO3 50 g
H2O deionised ad 1000 mL

Pre-culture-medium:
Stock solution I 10.0 mL
Stock solution II 1.0 mL
Stock solution III 1.0 mL
Stock solution IV 1.0 mL
H2O deionised ad 1000 mL

Nutrient-medium (10-fold concentrated):
Stock solution I 100 mL
Stock solution II 10 mL
Stock solution III 10 mL
Stock solution IV 10 mL
H2O deionised ad 1000 mL

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Specification:
Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae

Origin and culture:
The culture of Desmodesmus subspicatus was obtained in February 2014 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 7 °C. From the stock culture, a permanent culture was prepare From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
calculating of the cell number every 24 h

Test conditions

Test temperature:
20.4 - 22.6 °C
pH:
0 h: 7.8
72 h: 7.7
Details on test conditions:
Experimental conditions:
Date: 19. – 22. May 2015
Treatments tested: 1.0 / 3.2 / 10 / 32 / 100 mg/L
Number of replicates: 6 replicates for the control
3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Temperature: 20.4 – 22.6 °C
Lighting: 5300 Lux
Control: deionised water with nutrient medium and algae
Treatments: test solution and algae
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Biological results:

Parameter Value
72 h NOEC (Growth Rate) 3.2 mg/L
72 h NOEC (Yield) 3.2 mg/L
72 h LOEC (Growth Rate) 10 mg/L
72 h LOEC (Yield) 10 mg/L
72h ErC10 3.9 mg/L
72h EyC10 3.2 mg/L
72h ErC50 38 mg/L
72h EyC50 10 mg/L
Results with reference substance (positive control):
GLP study no. 14070902R301:

Biological Results of K2Cr2O7
Parameter Value 95%-confidence-interval Range of interlaboratory test (EU C.3)
72h ErC50 0.99 mg/L 0.94 – 1.1 mg/L 0.60 – 1.03 mg/L
72h EyC50 0.55 mg/L 0.52 – 0.58 mg/L none stated

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was performed using 5 concentrations ranging from 1 to 100 mg/L. For the estimation of the EC50s of the test item, the fits showed sufficient statistical correspondence of the data with the dose-response-equation.
At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC-MS. The correlation between nominal and measured concentrations was very good. Therefore, the determination of the results was based on the nominal concentrations.

The 72h-EC50s of potassium dichromate were determined in a separate reference test (GLP study no. 14070902R301). For the estimation of the 72h-EC50s of the positive control, the fits showed sufficient sta-tistical correspondence of the data with the dose-response-equation. The values were within the normal range of the laboratory.

All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome.
Executive summary:

One valid experiment was performed.

 

The study was performed using 5 concentrations ranging from 1 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the absorption and the cell density of the cultures determined by a cell counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the three highest treatments containing 10, 32 and 100 mg/L.

 

At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC-MS. The measured concentrations lay between 99 % and 100 % of the nominal concentration at the beginning of the test and between 97 % and 101 % of the nominal concentration at the end of the test. Therefore, the determination of the results was based on the nominal concentrations.

The 72h-EC50s of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the normal range of the laboratory.

The following results for the test item stabilized EMIM BF4 were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

3.2 mg/L

10 mg/L

3.9 mg/L

38 mg/L

Yield

3.2 mg/L

10 mg/L

3.2 mg/L

10 mg/L