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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 MAR 2005 to 24 MAR 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study compliant to GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
No test item analysis due to very poor water solubility of test item
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
No test item analysis due to very poor water solubility of test item
GLP compliance:
yes (incl. QA statement)
GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: nanoform, no surface treatment
Details on test material:
Name of test material (as cited in study report): Permanent-Rot FRR
Analytical purity: Purity of main component: 95.96 % (w/w) (comp. 1))

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
Stock solution: The solution (100 mg/L test item was weighed out) was prepared with dilution water one day prior to application.
Dispersion treatment: The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 µm (RC, SCHLEICHER & SCHUELL).
Limit concentration: Saturated solution (100 mg/L)
Due to the very poor water solubility of the test item, the filtrated saturated solution prepared at nominal 100 mg/L was used in the limit test.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test organism: Daphnia magna STRAUS (Clone 5)
Reason for the selection: Daphnia magna STRAUS belongs, in accordance with the study of the test organism guideline, to the suitable Daphnia types and are bred in the test facility.
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder: DR.U.NOACK-LABORATORIEN, 31157 Sarstedt, Germany
Culture: In 2-3 L glass vessels with approximatively 1.8 L culture medium, at 21 deg C (temperatures of 20 - 25 deg C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 µE*mE(-2)*sE(-1).
Culture medium: Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Feeding: At least 5x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10E(6) cells/mL.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:

Test conditions

266 mg CaCO3/L
Test temperature:
20.8 deg C (18-22 deg C, +/- 1 deg C)
Start: 7.72 End: 7.60 - 7.69
Start: 7.8 End: 7.83 - 7.94
Dissolved oxygen:
Start: 8.9 End: 8.09 - 8.14
Start: 8.69 End: 7.67 - 7.93
Nominal and measured concentrations:
Due to the very poor solubility of the test item no test item analysis was carried out.
Details on test conditions:
Test vessels: Glass beakers (5 cm ID x 8 cm H), 50 mL volume will be used.
Volume of the study medium: 20 mL

Dilution water: According to EC 92/69 L383A C.2. Annex
Component - Concentration [mg/L] in dilution water
KCL 5.76
NaHCO3 64.8
CaCl2*2 H2O 294
MgSO4*7 H2O 123

Number of study organisms per concentration and control: 20 animals, divided into 4 (2*) parallel samples, each with 5 (10*) animals
Number of animals per test vessel: 5 (10*)
Number of parallel samples per concentration: 4 (2*)
Age of test organisms: 2 - 24 h old
Acclimation: Ca. 2 h
Test temperature: 18 - 22 deg C, +/- 1 deg C constant
Illumination: Diffuse light, illumination range max. 20 µE*mE(-2)*sE(-1)
Photoperiod: 16/8 h light/dark cycle
Feeding: The daphnids were not fed during the study.

A static preliminary range finding test was conducted as limit test with the saturated solution (NON-GLP-state). The definitive study was performed as a limit test with saturated solution (100 mg/L) under static conditions to enable the determination of immobilisation after 24 and 48 hours.
2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h.
The daphnids were acclimated to the dilution water ca. 2 hours before test start. The daphnids were not fed during the study.
Control replicates with dilution water (test medium without test item) were tested under the same conditions as the test groups.
20 daphnids divided into 4 (2*) groups with 5 (10*) animals per test vessel will be used. In the definitive study the immobilisation of the daphnids was observed after 24 and 48 hours of exposure. Water parameter were measured at begin and end of the study.
The reference item was tested in five concentrations within the concentration range 0.58 to 5.8 mg/L.

*) The information in brackets refers to the test of the reference item

Reference substance (positive control):
Potassium dichromate p.a. (Merck) 100% purity, Batch No. K 28974764

Results and discussion

Effect concentrationsopen allclose all
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
Neither for the controls nor for the treatment replicates were any immobilzed daphnids observed.
Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC10- and EC50- with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures.
EC-values after 24 h of the reference item in mg/L:
EC10 : 1.51
EC50 : 1.84 (CI 1.74 - 1.95)
EC100 : 3.20
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline
38412 L 30.
Reported statistics and error estimates:
EC10- and EC-50-values were only calculated for the reference analysis item via probit according to WEBER (1986).
Calculation of the confidence intervals were carried out using standard procedures according to BREITIG & TÜMPLING (1982).

BREITIG G. & TÜMPLING W. (1982): Methoden der Wasseruntersuchungen Bd.II, VEB Gustav Fischer Verlag, Jena.
WEBER E. (1986): Grundriss der biologischen Statistik, 9. Auflage

Applicant's summary and conclusion

Validity criteria fulfilled:
In an acute immobilisation test to Daphnia magna according to OECD 202 no effects on daphnids were observed at the nominal limit concentration of 100 mg/L. Due to the very poor water solubility of the submission substance, a filtrated saturated solution prepared at 100 mg/L was used.
EC50 (48h) > 100 mg/L nominal concentration
NOEC (48h) = 100 mg/L nominal concentration
Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution# (100 mg/L) as limit concentration of the submission substance, was determined compliant to GLP according to OECD 202 (2004) and EC Directive 92/69/EC Method C.2 (1992) (reliability category 1). The limit test was conducted under static conditions over 48 h. 20 test organisms were exposed to the limit concentration and control. No test item analysis was carried out. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.84 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled. At the saturated solution (100 mg/L) of the test item no biologically significant effect was determined. Neither for the control nor for the treatament replicates any immobilized daphnids could be observed.

#) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6.