(ethyl acetoacetato-O1',O3)(pentane-2,4-dionato-O,O')[propane-1,3-diolato(2-)-O,O']titanium

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-02 to 2018-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

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 0.13 mg/L)
- Sampling method: water samples were taken from the control, solvent control and 5.0 mg/L test group, from fresh and old media throughout the 96 Hour test period. Duplicate sets of samples at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) were taken 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 (DMF) and the volume adjusted to 10 mL to give a 50 mg/mL solvent stock solution. The stock solution was inverted several times to ensure adequate mixing and homogeneity. An aliquot (2.0 mL) of this solvent stock solution was dispersed in 20 liters of test 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):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK

ACCLIMATION
- Acclimation period: acclimatized to test conditions from 25 September 2018 to 02 October 2018
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: commercial trout pellets
- Health during acclimation (any mortality observed): 1 mortality in the 7 days prior to the start of the test

- All other template details: Not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not reported

Hardness:

approximately 140 mg/L as CaCO3

Test temperature:

maintained at 14 °C during the study period

pH:

7.3 to 8.2 during the study period

Dissolved oxygen:

9.6 to 10.3 mg O2/L during the study period

Salinity:

not applicable

Conductivity:

not reported

Nominal and measured concentrations:

Nominal test concentrations (mg/L): 5.0.
Geometric mean measured test concentrations (mg/L): 0.13.

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. Tthe 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. The results obtained cannot be used to confirm the presence of test item in its parent form and are not stability indicating.

Samples were provided for chemical analysis both untreated, and following centrifugation at 40000 g for 30 minutes; giving an indication of the dissolved and hence bioavailable fraction present.
Results of analysis for bulk samples of test solutions without pre-treatment ranged between 117% and 118% of nominal in freshly prepared media and between 82% and 88% of nominal in the old media, confirming that the test media had been correctly formulated.

Analysis of the fresh test preparations at 0 hours showed a measured titanium concentration of 0.031 mg Ti/L in centrifuged samples of media. The analytical result obtained from the fresh test preparation at 72 hours (5.7 mg Ti/L) was considered to be anomalous. Analysis of the old test preparations at 24 and 96 hours showed a decline in measured to 0.0014 and 0.028 mg Ti/L respectively in centrifuged samples.

Given the decline in measured test concentrations observed it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.13 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass exposure vessels
- Material, size, headspace, fill volume: 25 to 30 liter glass exposure vessels containing 20 liters of test media
- Aeration: aerated via narrow bore glass tubes
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per solvent control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water for supply Zone ZDB13, dechlorinated by passage through an activated carbon filter and partly softened
- Metals: Typical Water Characteristics for Supply Zone ZDB13 normally contains the standard amount of mg 7.46 Al/L, 19.9 µg Fe/L, 0.02 mg Cu/L, 0.51 µg Pb/L and 3.1 µg Ni/L
- Chlorine: Typical Water Characteristics for Supply Zone ZDB13 normally contains around 0.32 mg/L
- Alkalinity: pH 7.66
- Hardness: approximately 140 mg/L as CaCO3
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the tes

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 hours light and 8 hours darkness and 20 minute dawn and dusk transition periods
- Light intensity: not reported

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Range finding study : not applicable

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: not reported
- Other biological observations: no abnormalities detected
- Mortality of control: no mortaliy in the control group. One fish was found outside of tank in the solvent control and was reported.
- Other adverse effects control:
- Abnormal responses: no abnormalities detected
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: nor reported
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

not reported

Sublethal observations / clinical signs:

Geometric Mean Measured Test Concentration (mg/L)	Mortality (%)	Sub-lethal effects
Control	0	No abnormalities detected
Solvent control	14*	No abnormalities detected
0.13	0	No abnormalities detected

*One fish found outside of tank

Validity criteria fulfilled:

yes

Conclusions:

- The test was considered to be valid given that none of the control or solvent control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was equal to or greater than 60% of ASV (6.1 mg O2/L at 15 °C) in the control, solvent control and test vessels.
- Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values of greater than 0.13 mg/L based on the geometric mean measured test concentration (derived from the theoretical titanium content). The No Observed Effect Concentration (NOEC) was 0.13 mg/L.

Description of key information

A study according to OECD 203 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

Fresh water fish

Fresh water fish

Effect concentration:

5 mg/L