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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

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
Qualifier:
according to
Guideline:
other: OECD Series on Testing and Assessment No. 23 (2000): Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Preparation of Calibration Solutions: The test item (75.90 mg) was dissolved in methanol and made up to the mark in a 50 mL volumetric flask to prepare a stock solution with a concentration of 1518 mg/L. A stock solution aliquot of 0.25 mL was diluted to 20 mL with methanol/water (1/1; v/v) to obtain a working solution with a concentration of 19.0 mg/L. Defined volumes of the stock and the working solutions were further diluted with methanol/water (1/1; v/v) to obtain calibration solutions of the test item in the range of 0.0949 to 30.4 mg/L. These solutions were used to calibrate the analytical system.
- Preparation of Spiked Recovery Solutions: To demonstrate the validity of the method, untreated test water was spiked with the test item. Therefore, the test item (119.62 mg) was dissolved in methanol and made up to the mark in a 50 mL volumetric flask to prepare a stock solution with a concentration of 2392 mg/L. Defined volumes of this stock solution were diluted with test water to obtain spiked samples with a concentration of 7.18 mg/L. These solutions were freshly prepared five-fold and subjected to the same treatment as a sample but without storage. In addition, test water without the test item was analyzed after sample preparation (analytical blank).
- Preparation of Test Samples: The diluted test and control samples were thawed at room temperature for 30 minutes and shaken manually to obtain homogeneous sample solutions for the sample analysis.
- Sample storage conditions before analysis: The samples were diluted with methanol by a factor of two before they were stored deep-frozen and protected from light until analysis was performed.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For preparation of the test medium, a dispersion of the test item with the loading rate of 100 mg/L was prepared. The dispersion was stirred for 24 hours to dissolve a maximum amount of the different components of the test item in the dispersion. Thereafter, the dispersion was filtered through a membrane filter (0.45 μm pore size) and the undiluted filtrate was tested as WAF. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Controls: test water without test item; for evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna STRAUS
- 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 the laboratory.
- Age at study initiation (mean and range, SD): 6-24h old and were not first brood progeny.
- Method of breeding: The clone of this species has been bred at the laboratory 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: Three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT and cultivated at Harlan Laboratories under standardised conditions or a mixture of this algal suspension and a commercial fish diet.

ACCLIMATION
- Acclimation period: not applicable, in-house breeding
- Acclimation conditions (same as test or not): breeding conditions same as test

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
none

Test conditions

Hardness:
2.5 mmol/L; 250 mg/L as CaCO3
Test temperature:
20°C
pH:
7.9-8.0
Dissolved oxygen:
8.2-8.6 mg/L
Nominal and measured concentrations:
Water accommodated fraction (WAF) with a loading rate of 100 mg/L was tested.
The analytically measured test item concentration (based on the analysis of the three main components of the test item) in the test medium was 8.78 mg/L at test start and 8.48 mg/L at test end.
The biological results were related to the mean measured test item concentration (8.6 mg/L) calculated as the arithmetic mean of the concentrations measured at the start and end of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker (the test vessels were labeled with the study number and all necessary additional information to ensure unique identification).
- Type (delete if not applicable): closed - the test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: The test was performed in 100-mL glass beakers filled with 50 mL of test medium. The volume of test solution provided for each daphnid was 10 mL.
- 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.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341 was used in the study.
- Alkalinity: 0.8 mg/L
- Ca/mg ratio: 4:1 (Na:K 10:1)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used.
- Light intensity: Light intensity during the light period was approximately between 485 and 640 Lux.
- Other: The daphnids were not fed during the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation after 24h and 48h

TEST CONCENTRATIONS
- Test concentrations: Water accommodated fraction (WAF) with a loading rate of 100 mg/L was tested. Higher loading rates of the test item were not tested in accordance with the test guidelines. The limit test was based on the results of a range-finding test and of pre-experiments to the water solubility and dosage of the test item (non-GLP).
The analytically measured test item concentration (based on the analysis of the three main components of the test item) in the test medium was 8.78 mg/L at test start and 8.48 mg/L at test end.
The biological results were related to the mean measured test item concentration (8.6 mg/L) calculated as the arithmetic mean of the concentrations measured at the start and end of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate is tested as a positive control twice a year, result of the latest positive control test in April 2011 (48-h EC50: 0.62 mg/L)showed that the sens. of test organisms was within the internal historical range (48-h EC50:0.43-1.1 mg/L).

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 8.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
>= 8.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 8.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 8.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
>= 8.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 8.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 8.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: none noted
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none noted; no remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test.
Results with reference substance (positive control):
- Results with reference substance potassium dichromate valid? yes
- EC50: 0.62 mg/L (48h)
- Other: internal historical range (48-hour EC50 from 2000 to 2011: 0.43-1.1 mg/L)
Reported statistics and error estimates:
95% confidence interval could not be determined as no immobilisation occurred.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Mean measured concentration (mg/L):
– 48-hour EC50 > 8.6
– 48-hour EC0 and 48-hour NOEC ≥ 8.6
– 48-hour EC100 > 8.6
The test item had no acute toxic effects on Daphnia magna up to its solubility limit in test water under the present test conditions.
Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test (GLP) according to the EU Method C.2 and OECD Guideline 202. A limit test was performed to demonstrate that a water accommodated fraction (WAF) of the test item has no toxic effect on Daphnia magna at the loading rate of 100 mg/L. Higher loading rates of the test item were not tested in accordance with the test guidelines. A control (test water without test item) was tested in parallel.

For preparation of the test medium, a dispersion of the test item with the loading rate of 100 mg/L was prepared. The dispersion was stirred for 24 hours to dissolve a maximum amount of the different components of the test item in the dispersion. Thereafter, the dispersion was filtered through a membrane filter (0.45 μm pore size) and the undiluted filtrate was tested as WAF. The preparation of the test medium was based on "OECD Series on Testing and Assessment No. 23 (2000): Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures".

The analytically measured test item concentration (based on the analysis of the three main components of the test item) in the test medium was 8.78 mg/L at test start and 8.48 mg/L at test end.

The biological results were related to the mean measured test item concentration (8.6 mg/L) calculated as the arithmetic mean of the concentrations measured at the start and end of the test.

In the control and at the mean measured test item concentration of 8.6 mg/L, no immobilised daphnids were determined during the test period of 48 hours.

The biological results can be summarized as follows:

Mean measured concentration (mg/L)

– 24-hour EC50 > 8.6

– 24-hour EC0 ≥ 8.6

– 24-hour EC100 > 8.6

– 48-hour EC50 > 8.6

– 48-hour EC0 and 48-hour NOEC ≥ 8.6

– 48-hour EC100 > 8.6

In conclusion, the test item had no acute toxic effects on Daphnia magna up to its solubility limit in test water under the present test conditions [it is important to recognise that the maximum achievable dissolved concentration of the substance in the test medium is not the same as the water solubility of the substance as determined by OECD Guideline 105 (as typically, the concentration is less). This is due to the fact that the water solubility measurements made for regulatory purposes are usually made in distilled water (pH=6-9; here 6.3) and not test media (pH=7-8; here: 7.9-8) and that differences in pH of the test media and distilled water affects the solubility].