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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-27 to 2018-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 16GWFA037
- Expiration date of the lot/batch: 01.04.2019
- Purity test date: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: as the test item instantly hydrolyses on addition to water and titanium dioxide is produced as one of the degradation products, it was not possible to identify if the titanium measured originated from the parent substance or the degradate, and therefore the results obtained cannot be used to confirm the presence of test item in its parent form and are not stability indicating

- All other template details: Not reported
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, solvent control and nominal test concentration 5.0 mg/L (geometric mena measured concentration 0.0073 mg/L)
- Sampling method: Samples were taken from the freshly prepared control and 5.0 mg/L test group from the bulk test preparation at 0 and 24 hours and from the old test media (pooled replicates) at 24. Duplicate sets of samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (500 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 50 mg/mL solvent stock solution. An aliquot (200 µL) of this 50 mg/mL solvent stock solution was dispersed in 2 liters of deionized reverse osmosis water with the aid of magnetic stirring for approximately 10 minutes to give the required test concentration of 5.0 mg/L .
- Controls: negative control and solvent control (100 µL/L of dimethylformamide).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Other relevant information: Information provided by the Sponsor indicated the test item was not readily soluble in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Preliminary media preparation trials indicated that a dissolved test item concentration of approximately 3.5 mg/L was obtained from a solvent spike method of preparation indicating this to be the limit of water solubility of this item under test conditions

- All other template details: Not reported
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Age at study initiation (mean and range, SD): less than 24 hours
- Source: in house laboratory cultures
- Feeding during test
- Food type: mixture of algal suspension (Desmodesmus subspicatus) and GEMMA Micro 300 suspension
- Frequency: daily

- All other template details: Not reported
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
not reported
Test temperature:
21 ±1 ºC
pH:
7.6 to 8.1
Dissolved oxygen:
7.6 to 8.9 mg O2/L
Salinity:
not applicable
Conductivity:
not reported
Nominal and measured concentrations:
Nominal test concentrations (mg/L): 5.0.
Geometric mean measured test concentrations (mg/L): 0.0073.

Despite extensive method development, a suitable substance specific method of analysis for this test item could not be developed. Consequently, verification of exposure concentrations was based on the measured levels of total dissolved titanium in the samples taken during the test, from which the measured levels of the test item were calculated based on its theoretical titanium content. However, as the test item instantly hydrolyses on addition to water and titanium dioxide is produced as one of the degradation products, it was not possible to identify if the titanium measured originated from the parent substance or the degradate, and therefore the results obtained cannot be used to confirm the presence of test item in its parent form and are not stability indicating.

Analysis of the fresh test preparations at 0 and 24 hours showed measured titanium concentrations of 0.0012 and 0.0019 mg Ti/L respectively; equivalent to test concentrations of 0.0086 and 0.014 mg/L respectively, based on its theoretical titanium concentration. Analysis of the old test preparations at 24 and 48 hours showed measured titanium concentrations had declined, to less than the limit of quantification (LOQ; determined to be 0.00051 mg Ti/L) and 0.0011 mg Ti/L respectively; equivalent to less than 0.0037 and 0.0080 mg/L respectively as test item. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentration of 0.0073 mg/L (based on its theoretical titanium concentration) in order to give a "worst case" analysis of the data.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Material, size, headspace, fill volume: 100 mL of test preparation
- Renewal rate of test solution (frequency/flow rate): 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water – ISO Medium
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Salinity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0 hours and 24 hours for the freshly prepared test preparation. 24 hours and 48 hours for the old test preaparation

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: between 200 and 1200 Lux

- All other template details: Not reported
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities:
- Observations on body length and weight:
- Other biological observations: No sub lethal effects of exposure were observed throughout the test
- Immobilisation of control: none of the control or solvent control daphnids showed immobilization
- Other adverse effects control: none of the control or solvent control daphnids sother signs of disease or stress
- Abnormal responses: none
Results with reference substance (positive control):
EC50 (24 hour) : 0.79 mg/L; 95% confidence limits 0.73 to 0.86 mg/L
EC50 (48 hour) : 0.75 mg/L; 95% confidence limits 0.56 to 1.0 mg/L

NOEC (24 hour): 0.56 mg/L
NOEC (48 hour): 0.56 mg/L
LOEC (24 hour): 1.0 mg/L
LOEC (48 hour): 1.0 mg/L

There was no immobilization in 20 daphnids exposed to a geometric mean measured test concentration of 0.0073 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

 Time (Hour)  EC50 (mg/L)  NOEC (mg/L)
 24  >0.0073  0.0073
 48  >0.0073  0.0073
Validity criteria fulfilled:
yes
Conclusions:
- The test was considered to be valid given that none of the control or solvent control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
- The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the geometric mean measured test concentration gave a 48 Hour EC50 value of greater than 0.0073 mg/L (based on theoretical titanium content). The NOEC was 0.0073 mg/L.

Description of key information

A study according to OECD 202 is available. A suitable substance specific method of analysis for the test item could not be developed in the study. Consequently, verification of exposure concentrations was based on the measured levels of total dissolved titanium in the samples taken during the test, from which the measured levels of the test item were calculated based on its theoretical titanium content. However, the analytical method applied in the studies is not considered appropriate for the test item, thus the nominal concentrations are used for the chemical safety assessment.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
5 mg/L

Additional information