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:
From 10-Jan-2011 to 03-Feb-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with international guidelines.

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
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: For the determination of the actual test item concentrations, duplicate samples were taken from each treatment at the test start and end of each test medium renewal period. The nominal test item concentrations of 1.1, 2.3, 5.0, 11, 23 and 50 mg/L were tested.
- Sampling method:Immediately after sampling, sodium dithionite solution with a nominal concentration of 10 mg/mL was added to each sample (100 μL per 10 mL of sample volume) in order to stabilize the latter for measurement. The samples were analyzed immediately after sampling.
- Sample storage conditions before analysis: no storage

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: At the start of each test medium renewal period, a stock solution with a nominal concentration of 100 mg/L was prepared by completely dissolving 100 mg (dosed amounts: 100.0 and 100.4 mg) of test item in 1000 mL of test water using intense stirring for 3 minutes at room temperature in the dark. This intensively mixed stock solution was used in a series of dilutions to prepare the test media of all test item concentrations. The test media were prepared just before introduction of the daphnids (i.e., start of the test and prior to the test medium renewal). As the test item is photodegradable, the preparation of the test media was performed as far as possible in the dark.
- Eluate:/
- Differential loading:/
- Controls: no special treatment
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):no vehicle
- Concentration of vehicle in test medium (stock solution and final test solution):/
- 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. At the three highest test concentrations (nominal 11 to 50 mg/L), the test media were already colored (purple) at the beginning of the test medium renewal periods. At the end of the renewal periods, the test media of the nominal concentrations of 2.3 to 50 mg/L were colored, at the three highest concentrations the intensity of the coloration had increased to dark purple.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: originally supplied by the University of Sheffield / UK in 1992.
- Age at study initiation (mean and range, SD): At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
- Weight at study initiation (mean and range, SD):/
- Length at study initiation (length definition, mean, range and SD):
- Valve height at study initiation, for shell deposition study (mean and range, SD):/
- Peripheral shell growth removed prior to test initiation:/
- Method of breeding: Since 1992, the clone has been bred at Harlan Laboratories 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. 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 during test: no

ACCLIMATION /

QUARANTINE /

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
250 mg/l as CaCO3
Test temperature:
The water temperature was maintained at 20-21 °C.
pH:
In consultation with the Sponsor, the pH of the test water was lowered to 6.0 (using 1 M hydrochloric acid) to increase the stability of the test item in the test water. At the beginning of the test medium renewal periods, pH values ranged between 6.1 and 6.4 after adjustment. At the end of the test medium renewal periods, the pH values of the test media were between 6.5 and 7.0.
Dissolved oxygen:
The dissolved oxygen concentrations in the test media and control were at least 8.2 mg/L.
Salinity:
/
Nominal and measured concentrations:
Nominal concentrations: 1.1, 2.3, 5.0, 11, 23 and 50 mg/L.
Measured concentrations (calculated as the arithmetic mean of the two geometric means of the concentrations measured at the start and end of the test medium renewal periods): 0.36 *, 0.75*, 1.9, 3.8, 8.0, 17 mg/L (*½ LOQ (0.128 mg/L) was used for the calculation of the mean measured concentration if the concentration at the end of the renewal period was
Details on test conditions:
TEST SYSTEM
- Test vessel,Type (open / closed), Material, size, headspace, fill volume: 250-mL glass beakers filled with 200 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.
- Type of flow-through (e.g. peristaltic or proportional diluter):/
- Renewal rate of test solution (frequency/flow rate):every 24H
- No. of organisms per vessel:5
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4
- No. of vessels per vehicle control (replicates):/
- Biomass loading rate:1 daphnia per 40 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341 was used in the study. It consisted of analytical grade salts dissolved in purified water
- Total organic carbon:/
- Particulate matter:/
- Metals:/
- Pesticides:/
- Chlorine:/
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1
- Conductivity:/
- Culture medium different from test medium:No
- Intervals of water quality measurement: At the start and end of each test medium renewal period, the pH values, dissolved oxygen concentrations and water temperature were determined in each treatment. The appearance of the test media was visually recorded at the same time.

OTHER TEST CONDITIONS
- Adjustment of pH:In consultation with the Sponsor, the pH of the test water was lowered to 6.0 (using 1 M hydrochloric acid) to increase the stability of the test item in the test water.
- Photoperiod:The test was performed as far as possible in the dark to avoid a photolytic degradation of the test item.
- Light intensity:/

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: between 2.09 and 2.2
- Justification for using less concentrations than requested by guideline:/
- Range finding study not reported in the report (not GLP), but results used for selection of the concentrations of the definitive study.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
3.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence interval
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.75
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
17 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities:
- Observations on body length and weight:/
- Other biological observations:No other symptoms than immobility were observed.
- Mortality of control: No
- Other adverse effects control:No
- Abnormal responses:At the mean measured test concentration of 1.9 mg/L, 10 daphnids were trapped at the water surface.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:/
- Effect concentrations exceeding solubility of substance in test medium:/
Results with reference substance (positive control):
- Results with reference substance : The result of the latest positive control test in October 2010 (48-hour EC50: 0.56 mg/L, study C97643) showed that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2010: 0.43-1.1 mg/L).

Any other information on results incl. tables

Nominal test item concentration (mg/L)

Mean initially measured concentration (mg/L)

No. of daphnids tested

Immobilized daphnids after 24 hours

Immobilized daphnids after 48 hours

 

 

 

No.

%

No.

%

Control

---

20

0

0

0

0

1,1

0,36

20

0

0

0

0

2,3

0,75

20

0

0

0

0

5

1,9

20

0

0

0 (10*)

0

11

3,8

20

13

65

13 (1*)

65

23

8

20

18

90

18

90

50

17

20

20

100

20

100

*Value in parenthesis: number of test animals with symptoms of toxicity (daphnids trapped at the water surface)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
in the control no daphnids showed immobilization or other signs of disease or stress; the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels
Executive summary:

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

The nominal test item concentrations of 1.1, 2.3, 5.0, 11, 23 and 50 mg/L were tested. Additionally, a control group was tested in parallel. Since the test item was not stable over the test medium renewal periods (24 hours), the reported biological results are based on the mean measured test item concentrations (calculated as the arithmetic mean of the two geometric means of the concentrations measured at the start and end of the test medium renewal periods). The measured concentrations were 0.36, 0.75, 1.9, 3.8, 8.0 and 17 mg/L. The biological test results were as follows:

48-hour EC50: 3.8 mg/L

48-hour NOEC: 0.75 mg/L

48-hour EC100: 17 mg/L