Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-30 to 2010-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study processed conform to guideline under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LCA08042
- Lot/batch No.: 0826000002
- Expiration date of the lot/batch: September 16, 2011
- Storage condition of test material: At room temperature (about 20°C), away from direct sunlight

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method:Treatment samples and control samples were thawed at 25 °C for 30 minutes and shaken manually to obtain homogeneous sample solutions. Aliquots of the final solutions were analyzed by HPLC with UV/VIS detection
- Sample storage conditions before analysis: The samples were stored deep-frozen and protected from light until analysis was performed.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Due to the low water solubility of the test item, a dispersion with the loading rate of 120 mg/L was prepared by dispersing 120.07 mg of the test item in 1000 mL of test water. The dispersion was supported by ultrasonic treatment for 45 minutes and intense stirring by a magnetic stirrer over 2 hours in the dark, to dissolve a maximum amount of the test item in the dispersion.
The stirring period of 2 hours was chosen according to the results of a pre-experiment (without GLP). After the stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.20 μm). The undiluted filtrate was used as the highest concentrated test medium and as stock solution for the preparation of the test media of lower test concentrations. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= start of exposure).
- Eluate: not applicable
- Differential loading: a dispersion with the loading rate of 120 mg/L was prepared
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No auxiliary solvent or emulsifier was used.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna Strauss
- Source: A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories.
- Age at study initiation (mean and range, SD): 6-24 hours
- Method of breeding: in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests
- Feeding during test: The daphnids were not fed during the test.

ACCLIMATION
- Acclimation period: Daphnids were bred in reconstituted water of the quality identical to the water quality used in the tests. Therefore, acclimation was deemed not neccesary.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).
- Feeding frequency: three times a week

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
no post-observation was performed

Test conditions

Hardness:
2.5 mmol/L
Test temperature:
21°C
pH:
At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.1 and 7.9.
Dissolved oxygen:
The dissolved oxygen concentrations in the test media and control were at least 8.0 mg/L.
Salinity:
Not particularly stated in the study report. Reconstituted test water according to ISO 6341 [1] was used in the study. It consisted of analytical grade salts dissolved in purified water. The detailed mineral composition of the reconstituted water is given in the original study report.
Nominal and measured concentrations:
nominal concentration: 110 mg/L
analytical (measured) concentration: 92-96 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL glass beakers filled with 50 mL of test medium
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- Renewal rate of test solution (frequency/flow rate): A semi-static test with test medium renewal after 24 hours was performed during the test period of 48 hours. After 24 hours, the test organisms were placed in clean test vessels with freshly prepared test medium.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 1 daphnid per 2 mL of water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341 was used in the study.
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1
- Culture medium different from test medium: no
- Preparation of test water: Due to the nonspecific solubility of the test item, a dispersion of the test item with the loading rate of 110 mg/L was continuously stirred at room temperature in the dark over 2 hours. Then, the dispersion was filtered. The undiluted filtrate of the dispersion was used as the test medium. Additionally, a control was tested in parallel. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000

OTHER TEST CONDITIONS
- Photoperiod and light intensity: A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was approximately between 520 and 680 Lux.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
In the control and at the loading rate of 110 mg/L, no immobilized test organisms were observed during the test period of 48 hours (Table 1).

Therefore, the 48-hour NOEC and the 48-hour EC0 were determined to be at least at the loading rate of 110 mg/L. The 48-hour NOEC and the 48 hour EC0 might even be higher, but loading rates higher than 110 mg/L were not tested. The 48-hour EC50 and the 48 hour EC100 were clearly higher than the loading rate of 110 mg/L.

In conclusion, the test item LCA08042 had no acute toxic effects on Daphnia magna up to 110 mg/L (nominal) in the test water under the present test conditions.

No remarkable observations were made concerning the appearance of the test medium. It was a clear solution throughout the entire test duration.
Results with reference substance (positive control):
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in September 2009 (48-hour EC50: 1.1 mg/L, study C63680) indicated that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2009: 0.53-1.1 mg/L).
Reported statistics and error estimates:
no statistics applied

Any other information on results incl. tables

Table 1: Effect of LCA08042 on the Mobility of Daphnia magna

Treatment / Loading rate

No. of
daphnids
tested

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
48 hours

(mg/L)

 

No.

%

No.

%

Control

20

0

0

0

0

110

20

0

0

0

0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item LCA08042 had no acute toxic effects on Daphnia magna up to 110 mg/L (nominal) in the test water under the present test conditions.
Executive summary:

The acute toxicity of the test item LCA08042 to Daphnia magna was determined in a 48‑hour semi-static test according to the EU Commission Directive 92/69/, Part C.2, the Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004) under GLP.

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water.

Due to the nonspecific solubility of the test item, a dispersion of the test item with the loading rate of 110 mg/L was continuously stirred at room temperature in the dark over 2 hours. Then, the dispersion was filtered. The undiluted filtrate of the dispersion was used as the test medium. Additionally, a control was tested in parallel. 

The measured concentrations of the test item in the test medium were between 94 and 96 mg/L at the start and at the end of each test medium renewal period. Consequently, the test water renewal ensured nearly constant concentration of the test item during the test and the reported biological results are related to the loading rate of the test item of 110 mg/L.  

In the control and at the loading rate of 110 mg/L no immobilized daphnids were determined during the test period of 48 hours. 

The biological test results were as follows:

 

 

Loading rate

(mg/L)

– 24-hour EC50:

>110

– 24-hour EC0:

110

– 24-hour EC100:

>110

– 48-hour EC50:

>110

– 48-hour EC0and 48-hour NOEC:

110

– 48-hour EC100:

>110

 In conclusion, the test item LCA08042 had no acute toxic effects on Daphnia magna up to 110 mg/L (nominal) in the test water under the present test conditions.